Professional Documents
Culture Documents
Associate Editor
Pavel Axentiev MS
Research Associate
Diana Swisher MA
Authors
Analytical
Maltodextrin Assay
Elan Sudberg
Alchemists Laboratories
Costa Mesa, CA
High Performance Thin Layer
Chromatography (HPTLC)
Judy Nichols
CAMAG USA
Wilmington, NC
High Performance Liquid
Chromatography (HPLC)
Paula N Brown PhD
British Columbia Institute of
Technology
Burnaby, British Columbia, Canada
Proton Nuclear Magnetic Resonance
Spectrometry (1H NMR)
John Edwards PhD
Process NMR Associates
Danbury, CT
History
Aviva Romm MD CPM Herbalist
Tufts School of Medicine
Boston, MA
Roy Upton RH DAyu
American Herbal Pharmacopoeia
Scotts Valley, CA
Botanical Identification
Sophie Neale PhD
Royal Botanic Garden
Edinburgh, UK
Macroscopic Identification
Lynette Casper BA
Planetary Herbals
Scotts Valley, CA
Microscopic Identification
Vaishali Joshi PhD
National Center for Natural
Products Research
University of Mississippi
University, MS
Prof Dr Reinhard Lnger
AGES PharmMed
Vienna, Austria
International Status
Josef Brinckmann
Traditional Medicinals
Sebastopol, CA
Reviewers
Ezra Bejar PhD
Herbalife
Los Angeles, CA
Anna Bozzi Nising
Office for Biotechnology, Nutrition
& Consumers at scienceindustries
Zurich, Switzerland
Kim Colson PhD
Bruker BioSpin Corp
Billerica, MA
Stefan Gafner PhD
Toms of Maine
Kennebunk, ME
Ferdinant Malan Du Preez BA (SA)
Capetown, South Africa
John S Haller Jr PhD
Department of History
Southern Illinois University
Carbondale, Illinois
ISBN: 1-929425-29-5
James Neal-Kababick
Flora Research Laboratories
Grants Pass, OR
Len E Newton PhD FLS
Kenyatta University
Nairobi, Kenya
Eike Reich PhD
CAMAG Laboratory
Muttenz, Switzerland
Tom Reynolds BSC MSc ARCS
Jodrell Laboratory
Royal Botanic Gardens, Kew
London, UK
Final Reviewers
Kristie M Adams PhD
United States Pharmacopeia
Rockville, MD
David Cutler PhD
Jodrell Laboratory
Royal Botanic Gardens, Kew
Richmond, Surrey, UK
Harry HS Fong PhD
Department of Medicinal Chemistry
and Pharmacognosy
University of Illinois at Chicago
Chicago, IL
Olwen M Grace PhD
Jodrell Laboratory
Royal Botanic Gardens, Kew
Richmond, Surrey, UK
ISSN: 1538-0297
Medical Disclaimer
Beau Barnett
Santa Cruz, CA
Statement of Nonendorsement
Reporting on the use of proprietary products reflects studies conducted with these and is not meant to be a product
endorsement.
Cover Photograph
Harvesting Aloe vera in Gonzales,
Mexico. Photograph courtesy of
Aloecorp Inc.
Ta b l e o f C o n t e n t s
A l o e Ve r a L e a f
A l o e Ve r a I n n e r L e a f
Juice
Nomenclature
Nomenclature
Introduction
2
2
History
Identification
43
Macroscopic Identification
Organoleptic Characterization
Identification
43
Definition
Botanical Nomenclature
Botanical Family
Definition
Common Names
43
Botanical Identification
Macroscopic Identification
Organoleptic Characterization
Microscopic Identification
Constituents
Maltodextrin Assay
High Performance Liquid Chromatography (HPLC)
Proton Nuclear Magnetic Resonance Spectrometry (1H NMR)
Limit Tests
Analytical
11
Sourcing
Sustainability
Cultivation
Harvest
Handling and Processing
Storage
Qualitative Differentiation
Adulterants
Preparations
IASC-Certified Juice Products
Constituents
Analytical
References
43
43
50
18
22
International Status
27
A l o e Ve r a L e a f J u i c e
Nomenclature
28
Definition
Identification
28
Macroscopic Identification
Organoleptic Characterization
28
28
29
Maltodextrin Assay
High Performance Liquid Chromatography (HPLC)
Proton Nuclear Magnetic Resonance Spectrometry (1H NMR)
Limit Tests
Introduction
The following monographs were developed to establish
guidelines for determining the identity, purity, and quality of
the crude leaves of Aloe vera, as well as aloe vera leaf juice
(made from entire leaf) and aloe vera inner leaf juice (made
from the colorless inner parenchyma) raw materials and
products. These standards are in alignment with the criteria
of the International Aloe Science Councils certification
program (IASC 2012).
The historical and contemporary uses of Aloe vera
encompass different articles of commerce derived from the
plant. The most widely used medicinal aloe preparation has
been a laxative prepared from the strongly bitter exudate
contained in the aloitic cells of the vascular bundle sheaths
of aloe leaves. The exudate (latex) is typically boiled to a
thickened consistency (inspissated) and dried. This is one of
the oldest continuous drugs in pharmacopoeial history and
is referred to by a variety of names, some of which may cause
confusion with other popular aloe leaf products that have
been introduced over the past few decades. Names applied
to the concentrated and dried aloe exudate include aloe
latex, aloe sap, aloe exudate, aloe gum, sometimes
aloe juice, and often simply aloe or aloes. These
preparations are characterized by the presence of phenolic
constituents, particularly aloin A and B (also known as
barbaloin) and chromone aloesin, which are largely
responsible for the laxative effects. Concerns regarding
these compounds as potential carcinogens have recently
been raised (CIR 2007; NTP 2011), resulting in limits
being established by independent organizations (e.g., the
International Aloe Science Council) and restrictions placed
on aloe vera products in some countries (e.g., Argentina,
Bolivia, Brazil). The International Aloe Science Council
(IASC) has developed a set of identity, purity, and quality
standards for aloe vera juice products (i.e., leaf juice and
inner leaf juice) as well as a voluntary certification program.
Products certified by IASC are specifically prepared in a
manner that limits the total amount of aloin A and B in
single-strength raw materials and finished products to 10 ppm
or less, ensures the presence of acetylated polysaccharides at
or above a minimum level ( 5% dry weight), and includes
assays to ensure the absence of specific adulterants. Thus,
the pharmaceutical preparations derived from the exudate,
inherently designed for short-term laxative use, must be
clearly distinguished from aloe vera leaf and inner leaf juice
preparations, especially those meeting IASC certification
criteria. Other species of Aloe (e.g., A. ferox) have also
been used in commercial juice products; however, these
species and products are not currently addressed by these
monographs or the IASC certification program.
Botanical Family
Xanthorrhoeaceae (alternatively placed in Aloaceae and
Asphodelaceae)
Pharmacopoeial Definition
Aloe vera leaf consists of the whole fresh leaves of Aloe vera
(L.) Burm. f., conforming to the methods of identification
provided.
Common Names
English:
History
The plant genus Aloe has a history of economic and
medicinal use that spans thousands of years and is the
source of some of the oldest known herbal medicines. The
name aloe comes from the Greek (alo), supposedly
derived, in its turn, from the Hebrew allal or the similar
Arabic word alloeh, both meaning bitter a tribute to the
taste of the leaf exudate (Park and Lee 2006). The name
aloe is also commonly applied to various products derived
from the plant.
The specific epithet vera in the botanical name Aloe
vera is Latin for truth (hence the common name true
aloe) and was first applied to the plant by Linnaeus (1753)
as the name of a variety within a species which he named
Aloe perfoliata. Fifteen years later, two other botanists
independently raised the variety to a full species level: Philip
Miller, in his book The Gardeners Dictionary, referred to
the plant as Aloe barbadensis the name which may have
been accepted as correct, had not Nicolaas Laurens Burman
named it Aloe vera in his Flora Indica published at least ten
days earlier (Newton 1979; Stafleu and Cowan 1976, 1981).
Publication precedent dictates that the earlier nomenclature
of Burman be accepted (McNeill et al. 2012), and thus the
Table 1 Historical timeline on the medicinal use of Aloe and Aloe vera
4000 BCE
Recorded in Ancient Egypt as a sanctuary plant of immortality and used as a funerary gift to the pharaohs.
2200 BCE
The earliest documented medicinal use of aloe is found on a Sumerian clay tablet.
1550 BCE
The Ebers Papyrus, one of the earliest medical works known, describes the healing benefits of aloe for both
internal and external conditions.
356323 BCE
Alexander the Great reportedly conquers the island of Socotra to secure the trade of Aloe perryi throughout
Asia.
Approximately 51 BCE
Referred to as the plant of Cleopatra by ancient Egyptians, allegedly regarding its use as a beauty aid.
27 BCE14 CE
Aloe vera is introduced into Greco-Roman medicine during the reign of Augustus.
1st century CE
Aloes are mentioned in the Holy Bible as the substance used with myrrh to anoint the body of Jesus.
4168
Dioscorides, in his De Materia Medica, writes the first in-depth report of the pharmacologic actions of aloe.
23129
Pliny reports on the use of aloe for leprous sores and, when topically applied, to reduce perspiration.
618907
Aloe vera is used medicinally in China, both topically, for dermatitis, and orally.
9 century
960-1279
The official materia medica of the Song Dynasty describes the use of whole ground leaf for the treatment of
sinusitis, fever, skin conditions, and seizures in children.
14th16th centuries
In Europe, aloe is considered a purgative and a topical treatment of wounds and various skin conditions.
1492
Christopher Columbus, upon setting sail for the New World, writes in his journal, All is well, aloe is on board.,
introducing aloe to the Americas.
16501742
Aloe vera is first imported to London and included as Barbados aloe in the London Pharmacopoeia (1650) and
in the London Dispensatory in 1742.
th
1650present
1753
1768
1810-1820
A variety of aloe preparations are entered into the Massachusetts and the United States Pharmacopeias.
1851
Edinburgh chemists Smith and Smith extract a cathartic principle from aloe and name it aloin.
1867
1912
1935
Collins and Collins report on the use of Aloe vera to treat radiation burns, stimulating modern research into the
potential benefits of Aloe vera for a variety of skin conditions.
1959
1975present
Present
Aloe vera products are approved for therapeutic purposes in Australia, Canada, India, and Korea, among other
countries.
2a.
2b.
Identification
Botanical Identification
Herbaceous perennial, succulent, erect, suckering freely to
form large colonies. Stem: Short, up to 30 cm, or absent.
Leaves: Spirally arranged as a clustered rosette, fleshy,
yellowish-green to glaucous (young plants may have white
spots), lanceolate, 4060 x 67 cm, with hardened pale
teeth, 2 mm long, 1020 mm apart, along the margin.
Inflorescence: Erect, to 1 m, with 13 branches; raceme
1040 cm. Flowers: Yellow to red*, perianth in 2 whorls, the
outer whorl fused for less than half of its length, cylindric or
slightly swollen below, glabrous, 2040 mm; pedicels stout,
approximately 6 mm long, subtended by bracts; bracts 10
mm; stamens 6; ovary superior, 3-celled, with many ovules
in each cell; anthers and styles slightly projecting from the
perianth. Fruit: A woody capsule with many black seeds
(Carter et al. 2011; Holmes and White 2002; Long and
Lakela 1971; Wood 1997).
The flower color of Aloe vera is reported as yellow in some botanical
literature but wild specimens can have both red and yellow flowers, as do
many other aloes (Wood 1997). Different color forms of Aloe vera available
commercially may therefore be due to this genetic heritage or the result of
hybridization with other species or cultivars.
Macroscopic Identification
The leaf of aloe vera can be described as consisting of two
major parts: the outer green rind and the colorless inner
leaf. The inner leaf, alternatively referred to as gel, pulp,
mucilage layer, aquiferous tissue, or mesophyll, is a
clear, transparent, colorless mass, taking most of the leafs
diameter, which consists of parenchymatous cells that
contain clear liquid constituting what is known as aloe vera
inner leaf juice (also see the accompanying Aloe Vera Inner
Leaf Juice monograph). Between the rind and the inner leaf,
a vascular layer can be distinguished, containing a series of
tubules (vascular bundles), which run along the full length
of the leaf.
Fresh Leaf
Surface view: Blade thick, succulent, bayonet-shaped,
narrowly-lanceolate, 3050 cm in length, 10 cm wide at
base, long-acuminate; color green to glaucous, with whitish
spots in younger plants; margin has pale, white to redddish
or light-brown teeth or spines (Bailey and Bailey 1976;
Culbreth 1917; WHO 1999). In harsher growing areas with
limited water supply, the leaves tend to grow thicker and
have more parenchymatous tissue.
Transverse section: Adaxial surface slightly concave; abaxial
American Herbal Pharmacopoeia Aloe Vera Leaf 2012
3a.
3b.
3c.
3d.
3e.
3f.
4a.
4b.
Mild, bland.
Aroma:
Faint, characteristic.
Texture:
Microscopic Identification
The leaf of aloe vera consists of the following tissues:
epidermis, chlorenchyma, vascular bundles, and colorless
inner parenchyma. The microscopic anatomy of Aloe leaves
is fairly constant regardless of the species. Some species may
be distinguished by microscopic analysis of the leaf surface
(Cutler 2004; Li et al. 2003).
Epidermis
In transverse section, the leaf epidermis on both the adaxial
and abaxial surfaces consists of a single, uniform layer of
cells, approximately 2540 m thick, covered with a waxy
thick cuticle, approximately 68 m. The epidermal cells
are arranged in parallel with the long axis of the leaves
and contain few chloroplasts. Both surfaces of epidermis
have sunken stomata, with guard cells surrounded by 45
epidermal cells.
Chlorenchyma
Just below the epidermis, there are usually 810 layers
(the number of layers can vary) of hexagonal-to-rounded
chloroplast-containing chlorenchyma cells, approximately
5060 m wide. Some idioblasts, with needle-shaped oxalate
crystals, are also present. Chlorenchyma in Aloe species is
not differentiated into spongy and palisade layers.
(continued page 11)
Table 2 Macroscopic characteristics of Aloe vera leaf compared to leaves of other Aloe species occurring in trade worldwide
Aloe vera
A. ferox
A. arborescens
A. perryi
40-60 cm long, 10 cm
broad at base
Up to 100 cm long, 15 cm
broad at base
Up to 35 cm long,
approximately 7.5 cm
wide
5a.
5b.
5c.
5d.
5e.
10
6a.
6b.
Sustainability
Vascular Layer
A single row of vascular bundles occurs between the
chlorenchyma and the colorless inner parenchyma, which
it encircles. Well-developed parenchymatous cells surround
the vascular bundles, forming a bundle sheath. Sieve tubes
and companion cells are narrow. The phloem is not very
distinct, and the xylem is composed of narrow vessels and
tracheids with annular and spiral thickenings. The yellow
exudate is secreted by clusters of thin-walled aloitic cells
forming a cap at the outer (phloem) pole of the vascular
bundles, as part of the bundle sheath.
Inner Parenchyma
Occupying the center of the leaf, the inner parenchyma
is composed of large parenchymatous cells, approximately
400-500 m in diameter, that lack chloroplasts and are filled
with polysaccharide-rich juice.
Commercial Sources
and Handling
There are numerous quality control issues associated with
aloe vera products, including differentiating between
closely related species, procuring appropriate raw material,
processing the leaves to reduce the content of the phenolic
compounds, preserving active constituents during processing
and storage, and identifying adulterants. Multiple procedures
have been developed to address these issues. For a detailed
presentation of aloe vera processing see He et al. (2005),
Ramachandra and Rao (2008), and Waller et al. (2004).
Sourcing
The preponderance of aloe vera is grown in Mexico,
followed by southern Texas and Florida in the United States.
Other sources include Argentina, Central America, China,
the Dominican Republic, India, North Africa, Thailand,
and Venezuela.
Cultivation
The natural habitats of Aloe vera are almost certainly
subtropical, and the plant grows best when supplied with an
excess of 50 cm of rain annually, in nitrogen-rich, alkaline
soil (Waller et al. 2004). The Rio Grand Valley of Texas
has been identified as an ideal growing region for aloe
vera. Because of its thick and shallow root system, aloe vera
requires well-drained soil and does not tolerate deep tillage
(Waller et al. 2004). Good drainage also helps to prevent
root rot, to which aloe vera is prone. While most species of
Aloe typically grow in sandy soils, aloe vera has also been
shown to grow well in tuff or basalt soils, with well-drained
substrates allowing for better growth (Saks and Ish-shalomGordon 1995). It can also tolerate saline soils, but the
salinity negatively affects biomass (Tawfik et al. 2001).
Aloe vera is reproduced asexually or by seed. Numerous
shoots (pups), developing around the base of mature plants,
may be separated and transplanted when 1520 cm (68
inches) high and will have fully mature leaves in 3 years.
The shoots should be removed from mother plants at
least twice annually to encourage larger leaf growth in the
parent plants. In vitro propagation has been reported to
be an effective way of reproduction, sufficient to meet the
demands of the growing aloe vera industry (Hashemabadi
and Kaviani 2008; Meyer and van Staden 1991).
Planting densities reportedly range 45006000 plants
per acre. Morton (1977) reports that to reach maximum
development, with a single mature leaf weighing 1 pound,
low content of latex, and plentiful pulp, aloe vera plants
should be grown in partial shade, irrigated in dry seasons,
American Herbal Pharmacopoeia Aloe Vera Leaf 2012
11
7a.
7b.
7c.
7d.
7e.
7f.
7g.
7h.
7i.
7j.
7k.
7l.
12
Harvest
Aloe vera leaves are generally ready for harvest after 3
years of age. Proper harvesting is a labor-intensive process.
Collection must be done with heavy gloves and while
13
aqueous formulations.
Following enzymatic treatment, the material is subjected
to a series of filtration steps to remove any remaining
rind particles and the undesired phenolic compounds.
Various modifications and proprietary variations of the
filtration process exist. The material is then passed through
a depulping machine, similar to that used for depulping of
citrus fruits. This is immediately followed by sterilization
(e.g., pasteurization), which is necessary to prevent
degradation of the acetylated polysaccharides by various
exogenous and endogenous microorganisms (Waller et
al. 2004). Further, to remove aloins A and B and other
phenolic compounds, decolorization with activated carbon
is employed. The resulting product is a clear fluid, which
is similar in organoleptic characteristics to inner leaf juice.
Aloe Vera Inner Leaf Juice
To obtain this type of product, the outer part of the leaf (the
rind) is separated from the clear inner leaf and discarded
prior to expressing the juice. This process, when properly
done, can minimize the presence of phenolic compounds
in the finished juice product.
In modern production, the leaves are initially subjected
to washing with an antimicrobial agent (e.g., hypochlorite)
to remove dirt and surface bacteria. Often the exudate is
then allowed to drain from the bottom portion of the leaf
(He et al. 2005). This is followed by culling, trimming, and
15
Qualitative Differentiation
When assessing quality of unprocessed aloe vera leaf
material, special attention should be paid to the structural
integrity of the leaves, as any damaged tissue (e.g., tip
necrosis), even in a minor portion of the leaves, can become
a site for microbial contamination, which will potentially
affect the quality of all later stage material.
A distinct characteristic of unprocessed aloe vera leaves
is their content of phenolic compounds (e.g., anthrones,
chromones, and anthraquinones). These constituents occur
exclusively in the vascular layer of the leaves and are absent
in the clear inner leaf. With safety concerns regarding these
compounds, the aloe vera leaf juice industry began using
activated charcoal in the process called decolorization.
This practice can consistently reduce the concentration
of phenolic compounds in finished aloe vera leaf juice
products. In the case of manual or mechanical separation of
the inner leaf, simply washing the inner leaf fillets prior to
further processing removes most of the phenolics. However,
due to inadequate techniques used, these compounds may
still occur in non-decolorized inner leaf juice products. In
IASC-certified products intended for oral consumption, the
total content of aloins A and B should be less than 10 ppm
(based on single strength of the raw material or finished
product). In aloe vera leaf juice products to be utilized
topically in cosmetic products, the limit is set as less than 50
ppm (CIR 2007).
Qualitative parameters that can be applied to aloe vera
leaf products include content of compounds of medicinal
interest. In this regard, acetylated polysaccharides have
been extensively researched and are considered some of
the most biologically active components in aloe vera leaf.
Currently, the only validated method for determination
of the content of this group of compounds in materials to
be used in consumer products is proton nuclear magnetic
resonance spectrometry (1H NMR), described in the
Analytical section of the Aloe Vera Leaf Juice monograph.
Certain processing procedures involved in manufacturing
of aloe vera leaf productsparticularly, pasteurization and
cellulase treatmentdirectly affect polysaccharide content.
A review of a limited number of commercial aloe vera leaf
products suggests that polysaccharide concentrations vary
widely, with some products containing as low as 1% dry
weight of acetylated polysaccharides (Bozzi et al. 2007). The
term overprocessed typically refers to materials that have
undergone prolonged pasteurization and/or uncontrolled
enzymatic treatment and demonstrate almost complete
lack of polysaccharides. Overprocessed juices can also be
distinguished by yellow discoloration due to caramelization
of sugars (even in decolorized material), changes in aroma,
and increased bitterness (Waller et al. 2004). Changes in
color typically occur with aloe juice preparations over time
and can be an indicator of relative freshness. As reported by
Chang et al. (2006), the juice color changes from whitish,
when fresh, to yellow to brown when exposed to high
temperatures or oxidation (see Figure 10c-g).
Several other small molecules can be used as additional
markers of quality of aloe vera leaf products. Acetic acid is
16
Adulterants
A variety of other Aloe species are traded commercially and
sometimes are not accurately declared as to proper species.
Products misrepresenting the species used are violative of
federal labeling laws. A number of Aloe species (e.g., A.
ferox, A. arborescens, and A. perryi) are readily differentiated
by the shape of the leaves (see Macroscopic Identification
and Table 2).
Maltodextrin is commonly used as a carrier during
spray-drying of aloe vera leaf juices in the manufacture
of powdered concentrates. For this purpose, the ratio of
maltodextrin to juice powder is typically 1:1, corresponding
to the concentration of 50% dry weight in the finished
product. Maltodextrin may also be added to artificially
enhance polysaccharide content and has historically been
one of the most common adulterants in aloe vera inner leaf
juice products. If not fully disclosed in labeling, maltodextrin
is considered an adulterant. For methods of detection and
quantitation of maltodextrin, see Maltodextrin Assay
and Quantitative Proton Nuclear Magnetic Resonance
Spectrometry (1H NMR), respectively, in the Analytical
section of the Aloe Vera Leaf Juice monograph.
Dilution is another issue of concern, as products
may contain added water that can decrease the efficacy
of the product. If undeclared, such products are out of
compliance with labeling regulations. IASC establishes
specific standards of product quality and potency.
Products labeled aloe vera inner leaf juice should
consist solely of the liquid from the inner leaf. Isocitrate
(isocitric acid) was established by IASC as a negative marker
for the inner leaf. According to IASC guidelines, aloe vera
leaf juice products that contain more than 5% dry weight
of isocitric acid should be labeled as aloe vera leaf juice,
not aloe vera inner leaf juice (see IASC-Certified Juice
Products below). For estimation of isocitrate content, see
the Quantitative Proton Nuclear Magnetic Resonance
Spectrometry (1H NMR) in the Analytical section of the
Aloe Vera Inner Leaf Juice monograph.
Preparations
For the traditional preparation of the inner leaf, the tip,
butt, and toothed margins of the leaf are cut off, and the
inner leaf fillet is separated from the green rind. The latter
is discarded. If the bitter principles of the exudate are
10a.
10b.
10c.
10d.
10e.
10f.
10g.
10h.
10i.
10j.
17
Certification requirement
Acetylated mannans
5% by dry weight
Glucose
Present
Aloin
Maltodextrin
Solids
Ash
40%
Isocitrate
Constituents
Anthrones and
anthraquinones*
Fatty Acids
Vitamins
Amino Acids
Minerals
Acetylated
glucomannan
Aloe-emodin
Capric acid
b-Carotene
Arginine
Potassium
Acidic galactan
Lauric acid
Vitamin B1
Aspartic acid
Sodium
Mannans
Isobarbaloin
Myristic acid
Vitamin B2
Glutamic acid
Copper
Glucomannans
7-Hydroxyaloin
Pentadecanoic acid
Vitamin B6
Serine
Zinc
Arabinogalactan
Homonataloin
Palmitic acid
Vitamin C
Histidine
Chromium
Arabinans
Chrysophanol
Margaric acid
Choline
Lysine
Selenium
Glucogalactomannan
Anthranol
Stearic acid
Vitamin D
Threonine
Aluminum
Cellulose
Chrysophanol glucoside
Palmitoleic acid
Vitamin E
(a-tocopherol)
Valine
Magnesium
Pectins
Aloesaponarin I & II
Hexadecadienoic acid
Folic acid
Methionine
Calcium
Polyuronide
Helminthosporin
Oleic acid
Vitamin K
Leucine
Manganese
Sugars
Tetrahydro-anthracene
glucoside
Linoleic acid
Niacinamide
Isoleucine
Chlorine
Glucose
Anthracene
Linolenic acid
Enzymes
Phenylalanine
Sulfur
Mannose
Emodin
Organic Acids
Amylase
Tryptophane
Iron
Arabinose
Chromones*
Malic acid
Oxidase
Histidine
Lignins
Rhamnose
Aloesin
Succinic acid
Catalase
Hydroxyproline
Lipids
Fructose
p-Coumaroylaloesin
Lactic acid
Lipase
Glutamine
Cholesterol
Sucrose
Isorabaichromone
p-Coumaric acid
Alkaline
phosphatase
Proline
Campesterol
Xylose
Feruloylaloesin
Salicylic acid
Cellulase
Alanine
Sitosterol
Glucuronic acid
Aloeresin A, B, C, & D
Uronic acid
Aliinase
Tyrosin
Triglycerides
Fucose
Aloesone
Uric acid
Glyoxalase
Cystine
Lupeol
Galacturonic acid
Flavonoids
Cinnamic acid
Lectins
Asparigine
Campherenol
Tannins
Quercetin
Fumaric acid
Aloctin I & II
Glycine
Saponins
* Anthrones, anthraquinones, and chromones comprise most of the contents of the exudate which is obtained from the vascular-bundle sheaths aloitic cells.
These compunds are also present in whole unfiltered leaf juice products.
Carbohydrates
A significant amount of the solid components of aloe
vera leaf juice is made up of carbohydrates, including
polysaccharides, simple sugars, and a small amount of
oligosaccharides. Waller et al. (2004) estimated carbohydrates
to comprise 25% of the solid fraction of aloe vera leaf juice.
After complete hydrolysis, which converts all carbohydrates
into simple sugars, mannose and glucose accounted for up
to 85% of total sugars in the inner leaf juice, with a glucose/
mannose ratio of 1:1.3 (Waller et al. 1978). While glucose
occurs mostly in the free form, mannose exists largely in a
polymeric form.
Monosaccharides
D-Glucose is universally reported as the primary soluble
sugar in aloe vera leaf, comprising about 95% of the total
American Herbal Pharmacopoeia Aloe Vera Leaf 2012
19
OH
10
OH
OH
HO
O
OH
OH
12a.
Lipids
The lipid content of aloe vera leaves ranges 2.71%5.13%
dry weight (Femenia et al. 1999). Several steroids were
reported, including -sitosterol, campesterol, cholesterol,
stigmasterol, lophenol, 24-methyl-lophenol, 24-ethyllophenol, cycloartanol, 24-methylene-cycloartanol, and
lupeol (Tanaka et al. 2006; Waller et al. 1978). Yamaguchi
et al. (1993) detected a variety of alkanes and fatty acids in
freeze-dried gel.
Phenolic Compounds
Anthrones, anthraquinones, and chromones are the major
types of phenolic compounds in aloe vera leaf exudate (Park
and Kwon 2006) and are removed from IASC-certified juices
by decolorization. Anthrone C-glycosides aloins A and B
(also known as barbaloin) account for 10%25% dry weight
of the exudate (Boudreau and Beland 2006). Aloins A and
B are diastereomers that can be easily separated by HPLC.
Aloin A has a 10S configuration at C-10 with a orientation
(Figure 12b), while aloin B is the 10R diastereomer with an
orientation. Chromones include aloesin (formerly called
aloeresin B) and aloeresin A, which, together with barbaloin,
are regarded as the most prominent constituents of aloe latex
(Park and Kwon 2006; Reynolds 2004). A number of other
anthrones, which are mostly derivatives of barbaloin, are
reported in aloe vera leaf exudate (e.g., see Okamura et al.
12b.
OH
Organic Acids
As much as 75% of the solids in aloe vera leaf and inner leaf
juices may consist of organic acids, metal ions, and chloride
(Waller et al. 2004). Malic acid is the most abundant organic
acid in aloe vera inner leaf juice, with a content of 11.1%
40.4% weight of the solid fraction (Jiao et al. 2010). Since
malic acid is prone to bacterial degradation to lactic acid by
Lactobacillus spp., content of malic acid can be used as a
marker for freshness and quality of aloe vera leaf and inner
leaf juices. IASC aloe vera leaf juice products commonly
contain lower malic acid levels than aloe vera inner leaf
juice products (Waller et al. 2004).
Citrate, isocitrate, and isocitrate lactone have been
found in higher amounts in the outer portion of aloe vera leaf
(Waller et al. 2004). Hence, the presence of high amounts
of isocitrate is used as a negative marker for aloe vera inner
leaf juice products. Citric acid can also be added to products
as an acidulant or preservative. Other organic acids that
may be present in the juices include quinic acid (Paez et
al. 2000), fumaric, succinic, and acetic acids. Fumaric acid
and succinic acid are products of the citric acid cycle in the
plant. If this cycle is allowed to continue during storage,
enzymatic degradation of the polysaccharides can occur,
resulting in the increase of organic acid concentrations.
Another important outcome of degradation of aloe vera
polysaccharides is their deacetylization, which results in
the production of acetic acid (Bozzi et al. 2007). Thus,
estimation of the levels of these compounds can be used to
assess processing and storage conditions.
Minerals
According to Rajasekaran et al. (2005), the following
elements occur in the gel layer (inner leaf) of aloe vera
leaf, in the order of decreasing concentrations: Fe, Mn, K,
Zn, V, Na, Mg, Cu, Cr, Ca, and Pb. Conversely, Ca2+ was
the most abundant metal ion in aloe vera gel (inner leaf
American Herbal Pharmacopoeia Aloe Vera Leaf 2012
21
A na ly t i ca l
The following methods are provided for the identification
and quality assessment of aloe vera leaf and aloe vera
leaf juices (liquid and dry). The high performance thin
layer chromatography (HPTLC) is predominantly used
to confirm the identity of aloe vera leaves used for the
manufacturing of juice products, including the detection of
the prominent alternate species in trade. The method can
also be used to establish the presence of exudate compounds
(such as aloins A and B). However, as shown in the
chromatograms provided, the HPTLC methodology is not
applicable for the identification of processed juice products.
The high performance liquid chromatography (HPLC)
method is primarily used for the quantitation of aloins A and
B in products and can be used to ensure conformity with
the IASC limits for these compounds. For screening of the
presence of maltodextrin, (e.g., to confirm its absence if not
stated in labeling), a simple and inexpensive Maltodextrin
Assay is provided. For detailed detection and quantitation of
the primary compounds of interest in aloe leaf and its juices
(i.e., acetylated polysaccharides), including for compliance
with IASC criteria, see Proton Nuclear Magnetic Resonance
Spectrometry (1H NMR) in the Analytical section of the
Aloe Vera Leaf Juice monograph.
Figure 13a HPTLC chromatogram of Aloe vera leaf and leaves of other Aloe species (UV 366 nm)
Discussion of the chromatogram
The standards aloin A and aloin B (lanes 1 and 2, respectively)
show zones of orange-brown fluorescence (Rf 0.43) and aloe
emodin (lane 2) shows a zone of yellow fluorescence (Rf 0.79). The
chromatograms obtained with the test solutions show in the lower
part (Rf 0.30) a light blue fluorescent zone, in the central part (Rf
Figure 13b HPTLC chromatogram of Aloe vera leaf and leaves of other Aloe species (UV 366 nm, derivatized)
Discussion of the chromatogram
The standards aloin A and aloin B (lanes 1 and 2, respectively)
show zones of yellow fluorescence (Rf 0.43) and aloe emodin
(lane 2) shows a zone of dark red fluorescence (Rf 0.79). The
chromatograms obtained with the test solutions show in the lower
part (Rf 0.30) a light blue fluorescent zone and in the central part
(Rf 0.43) a yellow fluorescent zone due to aloin. In some samples a
faint, diffuse zone of red fluorescence (Rf 0.79) due to aloe emodin
can be detected (this is detected more clearly in all of the samples
in the pre-derivatized UV 366 nm image above, 13a). A zone of red
fluorescence due to chlorophyll is seen at the solvent front (not
detectable in the inner leaf samples on lanes 12, 13 and 14).
23
Figure 13c HPTLC chromatogram of Aloe vera leaf and leaves of other Aloe species (white light, derivatized)
Discussion of the chromatogram
The standards aloin A and aloin B (lanes 1 and 2, respectively)
show brown zones (Rf 0.43) and aloe emodin (lane 2) shows a
red-brown zone (Rf 0.79). The chromatograms obtained with the
test solutions show in the central part (Rf 0.43) a brown zone due
to aloin and a faint, diffuse red-brown zone (Rf 0.79) due to aloe
Lane 1:
Lane 2:
Lane 3:
Lane 4:
Lane 5:
Lane 6:
Lane 7:
Lane 8:
Lane 9:
Lane 10:
Lane 11:
Lane 12:
Lane 13:
Lane 14:
Lane 15:
24
Aloin A
Aloe emodin, aloin B
Aloe vera fresh whole leaf
Aloe vera fresh outer leaf
Aloe vera fresh whole leaf
Aloe vera fresh whole leaf
Aloe vera fresh whole leaf
Aloe ferox fresh whole leaf
Aloe arborescens fresh whole leaf
Aloe vera fresh inner leaf
Aloe vera fresh inner leaf
Aloe vera fresh inner leaf
Aloe vera fresh inner leaf
Aloe ferox fresh inner leaf
Aloe arborescens fresh inner leaf
emodin. The Aloe vera and unknown Aloe test samples show in
visible light a violet-brown zone (Rf 0.39) just below the zone due
to aloin. The Aloe ferox and Aloe arborescens samples (lanes 8, 9,
14 and 15) are lacking this zone. The presence of this zone can be
used to differentiate Aloe vera from the Cape Aloes (Aloe ferox).
Figure 14a HPTLC chromatogram of Aloe vera leaf, leaves of other Aloe species, and Aloe spp. products (UV 366 nm)
Discussion of the chromatogram
The standards aloin A (lanes 1 and 22) and aloin B (lanes 2 and
23) show zones of orange-brown fluorescence (Rf 0.43) and aloe
emodin (lanes 3 and 24) shows a zone of yellow fluorescence (RF
0.79). The chromatograms obtained with most of the test solutions
show in the lower part (Rf 0.30) a light blue fluorescent zone.
This zone is not detected in the Aloe vera juice, tablets, or flakes
(lanes 11, 20, and 21, respectively). In the central part (Rf 0.43) an
orange-brown fluorescent zone due to aloin is detected in most
samples, except for some of the processed Aloe vera products
(juices, gel, tablets, and flakes on lanes 11, 12, 14, 20, and 21,
respectively). A faint, diffuse zone of yellow fluorescence (Rf 0.79)
due to aloe emodin is seen in most samples, except for some of
the processed Aloe vera products (juices, gel, tablets, flakes,
and powder on lanes 11, 12, 14, 19, 20, and 21, respectively), and
the Aloe ferox juice (lane 16). A zone of red fluorescence due to
chlorophyll is seen at the solvent front in the unprocessed leaf
samples, the Aloe ferox juice (lane 15), and the two Aloe vera leaf
extracts (lanes 17 and 18).
Figure 14b HPTLC chromatogram of Aloe vera leaf, leaves of other Aloe species, and Aloe spp. products (UV 366 nm, derivatized)
Discussion of the chromatogram
The standards aloin A (lanes 1 and 22) and aloin B (lanes 2 and
23) show zones of yellow fluorescence (Rf 0.43) and aloe emodin
(lanes 3 and 24) shows a zone of dark red fluorescence (Rf 0.79).
The chromatograms obtained with most of the test solutions show
in the lower part (Rf 0.30) a light blue fluorescent zone. This zone
is not detected in the Aloe vera juice and flakes (lanes 11 and
21, respectively). In the central part (Rf 0.43) a yellow fluorescent
zone due to aloin is detected in most samples, except for some
of the processed Aloe vera products (juices, tablets, and flakes
on lanes 11, 12, 20, and 21, respectively). A faint, diffuse zone of
25
Figure 14c HPTLC chromatogram of Aloe vera leaf, leaves of other Aloe species, and Aloe spp. products (white light, derivatized)
Discussion of the chromatogram
The standards aloin A (lanes 1 and 22) and aloin B (lanes 2 and 23)
show brown zones (Rf 0.43) and aloe emodin (lanes 3 and 24) show
a red-brown zone (Rf 0.79). The chromatograms obtained with the
test solutions show in the central part (Rf 0.43) a brown zone due
to aloin This zone is non-detectable in some of the processed Aloe
vera products (juices on lanes 11 and 12, tablets on lane 20, and
flakes on lane 21) or in the Aloe ferox juice (lane 16). Many of the
samples also show a faint, diffuse red-brown zone (Rf 0.79) due
to aloe emodin. Again, this zone is not detectable in some of the
processed Aloe vera products (juices on lanes 11 and 12, gel
on lane 14, powder on lane 19, tablets on lane 20, and flakes on
Lane 1:
Lane 2:
Lane 3:
Lane 4:
Lane 5:
Lane 6:
Lane 7:
Lane 8:
Lane 9:
Lane 10:
Lane 11:
Lane 12:
Lane 13:
Lane 14:
Lane 15:
26
Aloin A
Aloin B
Aloe emodin
Aloe vera fresh whole leaf
Aloe vera fresh outer leaf
Aloe vera fresh whole leaf
Aloe vera fresh whole leaf
Aloe vera fresh whole leaf
Aloe ferox fresh whole leaf
Aloe arborescens fresh whole leaf
Aloe vera leaf juice
Aloe vera inner leaf juice
Aloe vera inner leaf juice
Aloe vera inner leaf juice
Aloe ferox juice
lanes 21) or in the Aloe ferox juice (lane 16). The Aloe vera and
unknown Aloe fresh whole leaf test samples, Aloe vera gel (lane
13), and Aloe vera whole leaf powder (lane 19) show in visible light
a violet-brown zone (Rf 0.39) just below the zone due to aloin. This
zone is non-detectable in the other processed Aloe vera products.
The Aloe ferox and Aloe arborescens samples (lanes 8, 9, 14 and
15) are lacking this zone. The presence of this zone can be used to
differentiate Aloe vera from the Cape aloes (Aloe ferox).
International Status
United States
Cosmetic Product: Aloe vera leaf and inner leaf juice is
included in many aloe vera skin care products.
Food Additive: Aloe vera (cited as aloe) is approved as a
flavoring agent or in conjunction with flavors only.
Dietary Supplement Ingredient: Aloe vera products designed
for oral consumption can be labeled and marketed as a
dietary supplement product (USC 1994), requiring FDA
notification and substantiation to support structure/function
claim statements.
Australia
Gel: May be used as an active ingredient in Listed
medicines in the Australian Register of Therapeutic Goods
(ARTG) for supply in Australia. Specific Indications:
Aids the relief of sunburn, insect bites, chafing rashes and
irritation of other sensitive skin areas. For the first-aid care
of minor burns: cool the affected area with cold water or
ice first for a suitable period before applying gel. Medical
advice should be sought for the care of more serious burns
(TGA 2009a).
Juice and Juice Concentrate: Some aloe vera juice and juice
concentrate beverages are viewed as non-traditional foods
and not as novel foods (FSANZ 2009). There are some
listed medicines described as Aloe vera drinking gel or
as Aloe vera juice. Standard Indications: Aids digestion;
helps maintain healthy digestive function; aids, assists
or helps in the maintenance or improvement of general
wellbeing (TGA 2009b). Specific Indications: Acts as a
general tonic, helps to maintain healthy digestive function,
and has a cleansing effect on the bowel (TGA 2009c).
Canada
Leaf Gel (Inner Leaf Juice): Included as a Natural
Health Product (NHP) active ingredient, requiring premarketing authorization and product license for overthe-counter (OTC) human use. Quality: Must comply
with the minimum specifications outlined in the current
NHPD Compendium of Monographs (NHPD 2007b).
Pharmacopoeial standards currently accepted by the
NHPD are the British Pharmacopoeia (BP), European
Pharmacopoeia (PhEur) and United States Pharmacopeia
(USP). If no monograph exists in the currently accepted
pharmacopoeia, other internationally recognized standards
can be used such as World Health Organization (WHO)
(NHPD 2007a). Compendial Indications (Topical):
Preparations containing at least 10%70% leaf gel are (1)
traditionally used in herbal medicine to treat minor burns,
including sunburns; traditionally used to assist in wound
healing; and (2) traditionally used in herbal medicine to
assist healing of minor wounds such as cuts and burns, and
minor skin irritations (NHPD 2008).
European Community
Gel (Inner Leaf or Inner Leaf Juice): As of June 2012,
aloe vera gel was listed as a low priority in the inventory
of herbal substances for assessment by the European
American Herbal Pharmacopoeia Aloe Vera Leaf 2012
27
Nomenclature
Pharmacopoeial Definition
Aloe vera leaf juice consists of the liquid derived from the
entire leaves of Aloe vera (L.) Burm. f., or the dry powdered
concentrate of the liquid. The juice contains not less than
5% dry weight of acetylated polysaccharides, determined by
proton nuclear magnetic resonance spectrometry.
Commercial Sources
and Handling
Identification
Macroscopic Identification
Acetylated
polysaccharides
5% dry weight
Glucose
Present
Aloin A & B
Maltodextrin
Solids
Ash
40%
Note: The organoleptic characteristics of aloe vera leaf juice products can
vary considerably, depending on the processing techniques and additives
used.
28
Certification Requirement
Constituents
The solids content of aloe vera leaf juice varies depending
on the processing techniques. The solids fraction contains
mainly carbohydrates, organic acids, and mineral salts
(Waller et al. 2004), with most of the other components
removed during processing. The typical composition of
aloe vera leaf juice is provided in Table 2. For a more
detailed discussion of aloe vera polysaccharides and other
constituents, see the Aloe Vera Leaf monograph.
Acetylated mannan
5.36
Glucose
6.76
Malic acid
15.28
Lactic acid
0.09
Citric acid
9.29
Pyruvic acid
0.18
Sorbate
0.17
Benzoate
0.29
Isocitrate
6.06
Isocitrate lactone
3.40
Acetic acid
0.21
Succinic acid
0.00
Formic acid
0.00
Fumaric acid
0.00
Ethanol
0.00
Maltodextrin
0.00
1a.
A na ly t i ca l
1b.
Maltodextrin Assay
1a. Aloe vera leaf juice with and without maltodextrin (left to right:
0%, 10%, 25%, 50% weight of maltodextrin) prior to color
reaction with iodine.
1b. Aloe vera leaf juice with and without maltodextrin showing
color reaction after the addition of iodine.
Reagents
Iodine Solution: In 250-mL volume flask, dissolve 10 g
potassium iodide (KI) in 25 mL water. Add 3.175 g iodine
(I2) and dilute to 250 mL.
Sample Preparation
Liquid Samples: Add 10 mL of the liquid sample being
tested to an empty test vial.
Detection
Add 13 drops of Iodine Solution to the samples being
tested. Cap the vials and invert one time, or swirl.
Results
In the presence of maltodextrin, the solution will turn
medium- to dark-brown or purple to black, depending on
the amount of maltodextrin present (Figure 1).
29
water and methanol, add another 1 mL of 0.1% acetic acid solution in water,
and sonicate, vortex, and centrifuge the sample again prior to filtering and
aliquoting it to a HPLC vial.
Standards Preparation
Aloin A stock solution (0.1 mg/mL): Weigh 10.0 0.1 mg of
aloin A into a 100-mL volumetric flask, add approximately
80 mL of methanol, mix thoroughly, and fill to mark with
methanol.
Sample Preparation
Liquid Samples
Processed samples (e.g., decolorized aloe vera leaf juice):
Directly filter approximately 1 mL of the sample through 0.2
m PTFE into a HPLC vial for analysis.
Instrumentation
The HPLC system must be equipped with a diode-array
detector or UV detector capable of monitoring at 357
nm, sample injection system, pump capable of delivering
constant flow up to 600 bar, temperature-controlled column
compartment, and computing data processor. Equilibrate
the HPLC system with the mobile phase prior to analysis.
Reagents
Acetic acid, glacial: ACS reagent grade or equivalent.
Methanol: HPLC grade or equivalent.
Acetonitrile: HPLC grade or equivalent.
Water: HPLC grade or equivalent.
Mobile phase A: 0.1% volume acetic acid in water. Prepare
by adding 1 mL of glacial acetic acid to 999 mL of 0.2
m-filtered Nanopure water. Sonicate for 10 minutes.
Capsules
Combine and homogenize the contents of 20 capsules and
follow instructions for powders above.
Note: For samples that are highly concentrated or viscous, add 1 mL of 0.1%
acetic acid solution in water followed by adding 1 mL of 0.1% acetic acid in
methanol. Sonicate for 5 min three times, vortexing for 30 seconds in the
sonication intervals. Centrifuge at 5000 rpm (4500 g) for 3 min. If samples
are still concentrated or viscous after the 1:1 dilution of 0.1% acetic acid in
30
Standard label
(g/mL)
Volume of 25-g/
mL standard
(mL)
Volume of 5-g/
mL standard
(mL)
Volume of 1-g/
mL standard
(mL)
Volume of 0.2%
acetic acid in
water (mL)
Volume of
methanol (mL)
Std-50
0.5
0.5
Std-25
Std-10
Std-5
Std-1
Std-0.2
where:
tA and tB = the retention times of aloin A and aloin B,
respectively;
WA and WB = the widths of the peak bases of aloin A and
aloin B, respectively.
Mobile phase A, %
Mobile phase B, %
0.0
83
17
8.0
83
17
12.0
100
13.0
100
14.0
83
17
18.0
83
17
k = (tA t0) / t0
where:
tA = the retention time of aloin A;
t0 = the retention time of the void volume.
Tailing Factor (T):
The tailing factor should be less than 1, at 5% of peak
height. The formula to use is as follows:
Equation 3
T = W0.05 / 2 * a
where:
W0.05 = peak width at 5% of the peak height;
a
= distance from the leading edge of the peak to
the midpoint.
Signal-to-Noise Ratio (S/N):
The signal-to-noise ratio should be monitored for the 0.2
ppm standard. The noise is calculated by multiplying
the standard deviation of the linear regression of the
drift by 6 (closest range to the aloin A peak is manually
selected in the software). The formula to use is as
follows:
Equation 4
31
mAU
40
Aloin B
Aloin A
3a.
50 ppm QC standard
40
30
30
20
aloin A
aloin B
20
10
10
-10
10
min
10
3b.
Calculations
Obtain the integrated area (I) values of aloin A in the
calibration standards and aloins A and B in the analyzed
samples. Construct a plot of concentration (g/mL) of the
aloin A calibration standard (x-axis) against the individual
peak area (y-axis). Calculate the slope (m), intercept (b) and
correlation coefficient (R2) of the best-fit line.
The concentration of aloin A or aloin B in the prepared
sample is calculated by the following equation:
Equation 5
Cv = (Ialoin A/B b) / m
where:
Cv
= concentration of aloin A or B in the sample vial, g/
mL;
Ialoin A/B = integrated area of the aloin A or B peak in the
sample;
b
= intercept of the calibration curve;
m
= slope of the correlation curve.
Determine the concentration of total aloin (Ct, g/mL) in
the sample vial by adding individual aloin concentrations:
Equation 6
Ct = Cv of aloin A + Cv of aloin B
Cs = Ct * V1 * D / Ms
where:
Cs = concentration of total aloin in the original sample, g/
mg;
Ct = concentration of total aloin A and B in the sample vial,
g/mL, determined in the Equation 6 above;
V1 = volume of the prepared sample, mL (here 1 mL);
D = dilution factor if applicable;
Ms = mass of original sample, mg.
To determine total aloin concentration in single-strength
juice, first obtain the strength value of the juice, typically
stated on the label or in the documentation accompanying
the material, such as certificate of analysis (COA). Pure aloe
vera leaf juice powder is usually designated as having 100
strength, based on the standard value of the solids content in
32
30
20
10
aloin B
aloin A
10
C1x = Cs * Ds * S
where:
C1x = concentration of total aloin, ppm or g/mL, in singlestrength aloe vera leaf juice;
Cs = total aloin concentration in the sample, determined in
the Equation 7 above, g/mg;
Ds = dilution factor, based on the strength of the product,
estimated by the following equation:
Equation 9
Ds = 100 / x
where:
x = strength of the powdered concentrate stated in
the documentation accompanying the material;
S = standard concentration of juice solids in single-strength
aloe vera leaf juice = 10 mg/mL.
Aloe vera leaf juice can also be marketed as liquid
concentrates, e.g., 5, etc. For liquid material, first obtain
the strength value of the liquid and determine the total
content of aloins A and B in single-strength juice by the
following equation:
Equation 10
C1x = Ct * D * Ds
where:
C1x = total concentration of aloins, ppm or g/mL, in single-
t critical value
0.05
12.706
0.05
4.303
0.05
3.182
Equation 6, g/mL;
Equation 11
Ds = 1 / x
where:
x = strength of the liquid concentrate stated in the
documentation accompanying the material.
For replicate samples calculate the mean and standard
deviation/error. Report the total concentration of aloin
utilizing a 95% confidence interval as per the following
formula:
Equation 12
where:
= mean of replicates of the sample, calculated by the
following equation:
Equation 13
where:
n = number of replicates;
= t critical value, taken from Table 5;
= standard error of the mean, calculated by the
following equation:
Equation 14
where:
s = standard deviation;
n = number of replicates.
Representative chromatogram of aloins A and B standards
are shown in Figure 2. Representative chromatogram of aloe
vera leaf juice is shown in Figure 3.
33
Sample Preparation
Liquid Juice Samples and Aloe Vera-Containing
Commercial Products
Dissolve 150-200 mg of liquid aloe vera juice sample and
5-10 mg of the internal standard (nicotinamide) in ~0.7
mL deuterium oxide (D2O) and transfer into a 5-mm NMR
tube.
Freeze-Dried Juice Samples or Commercially Dried Juice
Products
Dissolve 20-50 mg of dried aloe vera leaf or inner leaf juice
powder and 5-10 mg of the internal standard (nicotinamide)
34
Note: The exact amount of sample or standard is not important, but weights
must be recorded to the nearest 0.1 mg. Volume of solvent is also not critical
as the final result will be calculated in terms of % weight and does not require
a volume to be used as is required for mg/mL calculations.
Reagents
NMR solvents
D2O (99.9% D-atom) + 0.01mg/ml 4,4-Dimethyl-4silapentane-1-sulfonic acid (DSS) (0.7 mL) (e.g., Cambridge
Isotope Laboratories, Andover, MA).
DCl (20% in D2O, 99.5% D-atom) (e.g., Cambridge Isotope
Laboratories, Andover, MA).
Note: Equivalent deuterated solvents from other manufacturers can be used.
Equipment
NMR spectrometer
Varian Mercury-300MVX with 1H-19F/15N-31P 5-mm PFG
AutoX DB Probe or 5-mm H/F/P/C 4-nucleus probe.
Operating with Varian VNMR-6.1C software.
Equivalent NMR systems and software include those
from the following manufacturers: Agilent/Varian (VNMR or
VNMRJ software), Bruker (Topspin software), JEOL (Delta
software). The necessary requirements are 1H Resonance
Frequency of 300-500 MHz and a functional 1H probe.
Examples of third party commercial and non-commercial
NMR software capable of processing spectral data acquired
on commercial NMR spectrometers (as above) include
ACD/NMR Processor (ACD/Labs), MNOVA (Mestrelab
Research), SpinWorks (freeware), Chenomx NMR Suite
(Chenomx).
Weighing equipment
Calibrated weighing balance capable of measuring
accurately to 0.1 mg.
Freeze dryer
Virtis BTK Benchtop K Manifold (SP Industries) or
equivalent.
Analytical Conditions
The typical NMR instrument parameters are shown in
Table 6. There is some variation of these parameters brought
about by differences in field strength and the sweep width
variations. All experiments must be optimally shimmed and
the acceptance criteria for acceptable spectral performance is
based on the quality of the nicotinamide standard resonance
located at 7.65 ppm which should optimally be a well
resolved, symmetric, 4 peak multiplet. The water resonance
set to 4.8 ppm is utilized as the chemical shift standard in
non-acidified samples. Preferentially internal chemical shift
standards readily available in NMR deuterated solvents DSS
or 3-(trimethylsilyl)-2,2,3,3-tetradeuteropropionic acid
(TMSP-d4) can also be utilized as the reference for 0 ppm.
3-8 seconds
2-6 seconds
Frequency, MHz
300-500 MHz
Nucleus
Number of Pulse Accumulations*
16-256
Zero-filled Points
32768-262144
Pulse sequence
Single pulse
Solvent
D2O
16
Signal-to-Noise ratio
(S/N) > 10
LOD, mg/mL
LOQ, mg/mL
Acetylated
mannan
< 0.05
< 0.1
Glucose
< 0.05
< 0.05
Malic acid
< 0.05
< 0.05
Lactic acid
< 0.005
< 0.005
Acetic acid
< 0.001
< 0.005
16384-84000
Line Broadening
Signal-to-Noise
ratio (S/N) > 3
Substance
Temperature
Ambient (25 C)
0.35 Hz
8
60 seconds
35
Chemical shift,
ppm
Acetylated
mannan
2.0-2.3
4.25
Malic acid
4.45
a-Glucose
4.6
b-Glucose
5.2
Isocitric
lactone
5.05
Isocitric acid
36
Equation 3
where:
CAP
WNic
IAcMann
NNic
MWAcMann
INic
NAcMann
MWNic
MWMann
MWGl
Wsample
where:
CAP
= content of acetylated polysaccharides in the sample,
% weight;
WNic = weight of added internal standard (nicotinamide),
mg;
IAcMann = integration area of acetylation methyls (multiple
peaks in 2.0-2.3 ppm region);
INic
= sum of integration areas of the 4 aromatic CH peaks
of the nicotinamide standard;
Wsample = weight of sample, mg.
Quantitation of Maltodextrin
According to IASC criteria, if maltodextrin is added to aloe
vera leaf juice during manufacturing, its content should be
accurately declared; otherwise, it is considered an adulterant.
Figure 7 shows an example of aloe vera leaf juice powdered
concentrate that contains 75% weight of maltodextrin and
25% weight of leaf juice powder. The peak at 5.4 ppm can
be used to determine the quantity of maltodextrin present.
Care must be taken to distinguish between the broad single
resonance of maltodextrin and the doublet that arises at the
Table 9 Chemical shift values, peak descriptions, and molar conversion factors that can be used for detection and quantitation of aloe vera
leaf juice preservatives, additives, and degradation products
Compound
Type of compound
Signal
Additive
1.1
1.15
Degradation product
1.33
Preservative
1.82
Acetic acid
Degradation product
1.96
Pyruvic acid
Degradation product
2.35
2.5-3.0
Degradation product
2.6
Glycerol
Additive
3.5
Glycine
Additive
3.51
Sucrose
Additive
5.4
Degradation product
6.5
Preservative
7.95
Degradation product
8.2-8.3
Propylene glycol
Ethanol
Lactic acid
Potassium sorbate
Citric acid
Succinic acid
Fumaric acid
Sodium benzoate
Formic acid
where:
where:
CMDX = content of maltodextrin in the sample, % weight;
WNic = weight of added internal standard, mg;
IMDX
INic
Wsample
where:
CX = concentration of the component, % weight;
WNic = weight of added internal standard (nicotinamide),
mg;
American Herbal Pharmacopoeia Aloe Vera Leaf Juice 2012
37
Malic Acid:
The malic acid CH signal at 4.3 ppm is usually present as a
multiplet but is often broadened due to chelation effects. In
some cases there is considerable complexation of malic acid
with other components in the juices. This can lead to broad
resonances for malic acid that are more difficult to properly
integrate and quantify, particularly in powdered aloe vera leaf
juice samples. The addition of a mineral acid (DCl) breaks
up the malic acid complexes and allows proper integration
of the malic acid CH signals (Figure 7). As stated previously,
the pH of the final solution is not important if the analysis
is to be performed by an analyst. However, the pH must be
known and controlled if automated spectral deconvolution
methods are to be used. Provided that the molar weight of
malic acid is 134.1 g/mol, and the resonance peak used for
estimation is CH (N = 1), the calculation is the following
(Equation 8):
where:
Glucose:
The anomeric proton signals of both the a (doublet at
5.2 ppm) and b (doublet at 4.6 ppm) anomers of glucose
are used to quantify the glucose content of aloe vera leaf
juice. The molar weight of glucose is 180.2 g/mol, and the
resonance groups used for quantitation are CH (N = 1). The
calculation is the following (Equation 9):
Equation 9
where:
CGlu = concentration of glucose in the sample, % weight;
mg;
= integration area of the CH proton resonance of
a-glucose (5.2 ppm);
= integration area of the CH proton resonance of
b-glucose (4.6 ppm);
= sum of integration areas of the 4 aromatic CH
peaks of the nicotinamide standard;
= weight of sample, mg.
Isocitrate:
The instructions for isocitrate quantitation are provided in
the Aloe Vera Inner Leaf Juice monograph.
Isocitrate Lactone:
The CH resonance (doublet at 5.05 ppm) of the isocitrate
lactone molecule (176.0 g/mol) is utilized for quantitation.
The final formula is the following:
Equation 10
where:
CICL
WNic
39
Figure 6 1H-NMR spectrum of a 5x aloe vera leaf juice analyzed without freeze-drying
Discussion
This spectrum shows a 5x aloe vera leaf juice with benzoate and
sorbate added as preservatives. All signals typically observed in
freeze-dried 5x leaf juice are observed, but at a lower intensity.
A large H2O resonance is observed at 4.8 ppm. The spectrum
shows some degradation products, such as acetic acid (singlet
at 1.98 ppm) and lactic acid (doublet at 1.3 ppm), and signs of
IICL
INic
Wsample
Citrate:
Citrate (192.0 g/mol) CH2 resonances appear in the 2.5-3.0
ppm region and are overlapped by the CH2 resonances of
malic acid, isocitrate, and isocitrate lactone. These latter
components can be quantified from their CH resonances
found in the 4.25-5.05 ppm region. Thus, once the integrated
values for the CH protons of malic acid (IMA), isocitric acid
(IICA), and isocitrate lactone (IICL) have been obtained, it is
possible to obtain the molar integration value of citric acid
(ICA / NCA) by subtracting the intensity of these CH carbons
(multiplied by 2 to adjust for the CH2 signal intensities
being subtracted for these molecules) from the CH2 region,
and then dividing by the four citric acid protons (NCA) that
are present in that region. The final calculation is shown in
Equation 11.
40
Equation 11
where:
CCA = concentration of citric acid in the sample, % weight;
WNic = weight of added internal standard (nicotinamide), mg;
ICA
where:
I2.5-3.0 = total integration area of the 2.5-3.0 ppm region;
INic
= sum of integration areas of the 4 aromatic CH peaks
of the nicotinamide standard;
Wsample = weight of sample, mg.
Degradation Products:
The presence of degradation products can be used to assess
the quality of the material and give indications of sample
processing and storage conditions. Chemical shift values,
peak descriptions, and molar conversion factors for typical
degradation products of aloe vera acetylated polysaccharides
can be found in Table 9.
Figure 7 1H-NMR spectra of a commercial aloe vera leaf juice powder containing 75% dry weight maltodextrin, before and after acidification
with 1 drop of 20% DCl in D2O
Discussion
The region around 2.8-3.0 shows the presence of isocitrate in the
non-acidified sample (lower spectrum). Acidification with 1 drop
Limit Tests
Solids Content: Not less than 1.0% in single-strength (1)
juice, determined by drying a 10-gram
sample of the liquid at 105 C for 24 hours
(Wang and Strong 1995).
41
Figure 8 1H-NMR spectrum of a commercial, freeze-dried 100x aloe vera leaf juice powder
Discussion
42
Identification
Macroscopic Identification
Aloe vera inner leaf juice is a viscous, cloudy to transparent
liquid.
Constituents
Organoleptic Characterization
A na ly t i ca l
Liquid (single-strength)
Color: Clear to caramel-colored.
Aroma: Odorless to mildly vegetative.
Taste: Tasteless to slightly bitter.
Maltodextrin Assay
Commercial Sources
and Handling
43
Certification Requirement
Component
Acetylated mannan
8.98
Glucose
12.89
Present
Malic acid
23.16
Lactic acid
0.06
Citric acid
5.47
Pyruvic acid
0.04
Maltodextrin
Sorbate
0.73
Benzoate
0.53
Solids
Isocitrate
0.00
Ash
40%
Isocitrate lactone
0.00
Acetic acid
0.27
Isocitrate
5% dry weight
Succinic acid
0.00
Formic acid
0.00
Fumaric acid
0.00
Ethanol
0.15
Maltodextrin
0.00
Acetylated
polysaccharides
5% dry weight
Glucose
Aloin
Cv = (Ialoin A/B b) / m
where:
Cv
= concentration of aloin A or B in the sample vial, g/
mL;
Ialoin A/B = integrated area of the aloin A or B peak in the
sample;
b
= intercept of the calibration curve;
m
= slope of the correlation curve.
Determine the concentration of total aloin (Ct, g/mL) in
the sample vial by adding individual aloin concentrations:
Equation 2
Ct = Cv of aloin A + Cv of aloin B
Cs = Ct * V1 * D / Ms
C1x = Cs * Ds * S
where:
C1x = concentration of total aloin, ppm or g/mL, in singlestrength aloe vera inner leaf juice;
Cs = total aloin concentration in the sample, determined in
the Equation 3 above, g/mg;
Ds = dilution factor, based on the strength of the product,
estimated by the following equation:
where:
Cs = concentration of total aloin in the original sample, g/
mg;
Ct = concentration of total aloin A and B in the sample vial,
g/mL, determined in the Equation 2 above;
V1 = volume of the prepared sample, mL (here 1 mL);
D = dilution factor if applicable;
Ms = mass of original sample, mg.
Equation 5
44
Ds = 200 / x
where:
x = strength of the powdered concentrate stated in
the documentation accompanying the material;
S = standard concentration of juice solids in single-strength
aloe vera leaf juice = 5 mg/mL.
t critical value
0.05
12.706
0.05
4.303
0.05
3.182
C1x = Ct * D * Ds
where:
C1x = total concentration of aloins, ppm or g/mL, in singlestrength aloe vera inner leaf juice;
Ct = total aloin concentration in the sample vial from
Equation 2, g/mL;
D = dilution factor if applicable;
Ds = dilution factor, based on the strength of the product,
estimated by the following equation:
Equation 7
Ds = 1 / x
where:
x = strength of the liquid concentrate stated in the
documentation accompanying the material.
For replicate samples calculate the mean and standard
deviation/error. Report the total concentration of aloin
utilizing a 95% confidence interval as per the following
formula:
Equation 8
where:
= mean of replicates of the sample, calculated by the
following equation:
Equation 9
where:
n = number of replicates;
= t critical value, taken from Table 3;
= standard error of the mean, calculated by the
following equation:
Equation 10
where:
s = standard deviation;
n = number of replicates.
45
CICA = (WNic * IICA * NNic * MWICA) / (INic * NICA * MWNic * Wsample) * 100%
where:
CICA = content of isocitrate in the sample, % weight;
46
NNic
MWICA
INic
NICA
MWNic
Wsample
Equation 2
CICA
where:
CICA
= content of isocitrate in the sample, % weight;
WNic = weight of added nicotinamide internal standard,
mg;
IICA
= integration area of CH proton resonance of
isocitrate (doublet at 4.45 ppm (dissolved in D2O
and acidified with DCl), or 4.2 ppm (dissolved in
D2O));
INic = sum of integration areas of the 4 aromatic CH peaks
of the nicotinamide standard;
Wsample = weight of sample, mg.
Limit Tests
Solids Content: Not less than 0.5% in single-strength (1)
juice, determined by drying a 10-gram
sample of the liquid at 105 C for 24 hours
(Wang and Strong 1995).
47
48
a single drop of 20% DCl in D2O, shifts the malic acid, citrate,
isocitrate, and isocitrate lactone signals (as well as formic acid,
acetic acid, succinic acid, and the nicotinamide standard signals),
increasing the resolution of the malic acid/citrate/isocitrate CH2
region (2.4-3.0 ppm). Acidification can also cause the malic acid CH
resonance to overlap with the b-glucose resonance. The isocitrate
CH resonance, which is of main interest in this part of the analysis,
is typically found as a discrete doublet not overlapped with any
other signals, as indicated at 4.5 ppm. All other components are
calculated from the spectrum before acidification.
49
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