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ISSN 0975-6299

Vol.1/Issue-4/Oct-Dec.2010

International Journal of Pharma and Bio Sciences VARIATIONS IN VALUES OF PROXIMATE ANALYSIS IN AERVA LANATA JUSS EX SCHULTES, HEDYOTIS CORYMBOSA (L.) LAM. AND LEPTADENIA RETICULATA (RETZ.) W. & A. MAMMEN D1*, DANIEL M2 AND SANE RT3
1* 2

Department of Chemistry, M.S. University of Baroda, Vadodara-390 002,India. Department of Botany, M.S. University of Baroda, Vadodara-390 002, India. 3 Guru Nanak Institute for Research and Development, G.N. Khalsa College, Matunga, Mumbai -400 019,India.
*Corresponding Author

drdenni.mammen@gmail.com

ABSTRACT
Quality control methods are of utmost importance for maintenance of quality of herbal medicines. Literature values are available for a number of medicinal plants. Such values are considered to be standard and at times taken for granted. The present work was carried out to determine if the values of proximate analysis are consistent even with change of season or region of collection. If the values vary, then it is of utmost importance to determine the best time and place of collection for the plant. The various parameters studied were ash analysis, extractive values and moisture content for three plants Aerva lanata, Hedyotis corymbosa and Leptadenia reticulata. Each plant was collected during summer, monsoon and winter to study the effect of change of season on the proximate analysis values. Similarly the analysis was carried out for samples collected from Gujarat, Maharashtra and Kerala, to study the effect of geographical variation in the plants. Interestingly, the values were found to be change with season and region of collection of these plants.

KEYWORDS
Aerva lanata, Hedyotis corymbosa, Leptadenia reticulata, proximate analysis, ash analysis, extractive values, variation.

INTRODUCTION
Quality controls of synthetic drug offer no problems with very well defined parameters of analysis. In contrast, herbal products represent a number of unique problems when quality aspects are considered. These are because of the nature of the herbal ingredients present therein, which are complex mixtures of different secondary metabolites that can vary considerably depending on environmental and genetic factors. Furthermore, the constituents responsible for the claimed therapeutic effects are frequently unknown or only partly explained. These complex positions of quality
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aspects of herbal drugs are further complicated by the use of combination of herbal ingredients as are being used in traditional practice. It is not uncommon to have as many as five or more different herbal ingredients in one product. Thus batch to batch variation starts from the collection of raw material itself in the absence of any reference standard for identification. These variations multiply during storage and further processing1. In olden times, vaidyas used to treat patients on individual basis, and prepare drug
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according to the requirement of the patient. But the scenario has changed now; herbal medicines are being manufactured on the large scale in pharmaceutical units worth US 60 billion dollars. The Indian manufacturers have to stress on various standardization processes to establish their products in the global markets. Starting from production of quality materials, analysis of raw materials for authentication, presence of foreign matter, organoleptic evaluation, microscopic examination, extractive values, chromatographic profiles, pesticide residues, heavy metal detection etc. are necessary for standardization of drugs. Similarly, the standardization methods of medicinal plants and their extracts have great importance in the fields of cosmetics and nutraceuticals, which are emerging as two most important segments in the global market. In order to step up quality in production and products, research and documentation are mandatory to supply to international requirements. This could be met with by referring global standards and international pharmacopoeias like Herbal British Pharmacopoeia, Chinese and Japanese Pharmacopoeia, Ayurvedic Pharmacopoeia, Ayurvedic Formulary of India and WHO Guidelines on Herbal Medicines. Accounting to WHO, quality control is the process involving the physicochemical evaluation of crude drug covering the aspects, such as selection and handling of crude material, safety, efficacy and stability assessment of finished product, documentation of safety and risk based on experience, provision of product information to consumer and product promotion. The procedures involve Macroscopic and Microscopic Examination, Foreign Organic Matter, Ash Values, Moisture Content, Extractive Values, Crude Fibre, Qualitative Chemical Evaluation, Chromatographic Examination, Qualitative Chemical Evaluation and Toxicological Studies3. The ash content of a crude drug is the inorganic residue remaining after incineration. It includes not only the inorganic salts, e.g. Calcium oxalate, occurring naturally in the drug; but also inorganic matter from external sources. The official ash value is an important tool to ensure the absence of an undue
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proportion of extraneous mineral matter introduced accidently or by design due to the type of manufacturing process used, e.g. earth, sand, floor sweepings etc. and to detect adulteration of the drug and adulteration by material of construction of manufacturing equipment, e.g. brick lined surface etc4. Four types of ash values used in routine pharmaceutical analysis are Total ash content, Acid insoluble ash, Water soluble ash and Sulphated ash. Total ash content - A figure for total ash content is useful when the contamination with calcium oxalate is very little. If more quantity of calcium oxalate is present, then the value for the acid insoluble ash is a better criterion of purity. Acid insoluble ash -Crude drugs containing larger quantity of calcium oxalate, can give variable results depending upon the conditions of ignition. Treatment of the ash with Hydrochloric acid leaves virtually only silica. Hence acid insoluble ash forms a better test to detect and limit excess of soil present as an impurity in the drug, than does the total ash. It is necessary to use ash-less filter paper during filtration and subsequent incineration, to limit the error. Water soluble ash -It is a measure of detection of water soluble impurities in drug or raw material. Sulphated ash -The determination of sulphated ash is widely used to control the extent of contamination by nonvolatile inorganic impurities in organic substances. For sulphated ash, the substance is ignited using small quantities of sulphuric acid, which decomposes and oxidises the organic matter, leaving a residue of inorganic sulphates. Reproducible results are readily obtained in this determination than in total ash determination, due to the higher stability of metal sulphates. Ash values, extractive values and moisture content for many medicinal plants are available in literature. These values are however, seldom reproducible at all places when the analysis is repeated. This may be attributed to the fact that season and place of collection cause a significant difference in the values obtained during previous analysis, and those reported in literature. Looking into all these problems the present study is undertaken wherein the seasonal and geographic variations in proximate analysis
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values of three important Indian medicinal plants Aerva lanata Juss ex Schultes, Hedyotis corymbosa (L.) Lam., and Leptadenia reticulata (Retz.) W. & A. were conducted. Aerva lanata (Bhadra in Sanskrit) is a common weed found in all plains districts and upto 900 metres elevation. It is widespread in the drier parts of the tropics and subtropics of Africa and Asia5. Plant decoction is diuretic and is given to remove bladder and kidney stones. It is also used to treat diarrhoea6. Leaf paste is mixed with gingelly oil and given to treat piles. Leaf and root paste is applied to treat pimples and skin infections. Leaf decoction is given as anthelmintic and demulcent. Root and flower decoction is given to treat headache. Root decoction is used as an antidote for snakebite. Root powder is used as tooth paste to treat toothache7. Whole plant is used to treat cough, boils, lithiasis (calculus formed by inorganic salts) and pus8. Hedyotis corymbosa (Parpataka) is a spreading, suffruticose annual, frequently found especially during monsoon in fields throughout India, Sri Lanka, tropical East Asia to Java and the Phillipines9. The plant is known to clear heat and toxins, activate blood circulation, promote diuresis and relieve stranguria (urinary obstruction). It is also known to act against tumours of the digestive tract, lymphosarcoma, as well as carcinoma of the liver and larynx. It is also active against

appendicitis, hepatitis, pneumonia, cholecystesis, urinary infection, cellulites and snake bite. Chinese folk medicine describes the plant to treat skin sores, ulcers, sore throat, bronchitis, gynaecologic infections and pelvic inflammatory diseases10-13. It is usually administered in the form of a decoction in remittent fever with gastric irritability and nervous depression caused by deranged bile. It is also used to treat jaundice and diseases of liver. The juice of the plant is applied to palms and soles to relieve the burning sensation during fevers. The plant is used as an anthelmintic. In Philippines the plant is boiled in water and the brew is used as a mouthwash for relief during toothache9. Leptadenia reticulata (Jivanti) is a twining shrub found in Gujarat, Sub-Himalayan tracts from Punjab to Sikkim and Khasi hills and throughout peninsular India, ascending up to an altitude of 900 metres. It is also distributed in tropical and sub-tropical parts of Asia and Africa9. It is considered to be a Rasayana (tonic) and is thus used to vitalize, nourish and rejuvenate the body. Its use as a galactogogue is well known. It is also used to cure weakness, chicken pox, dysentry, tuberculosis, cough, respiratory disorders, eye diseases and night blindness, as well as certain skin diseases5-6.

EXPERIMENTAL
The parameters looked into in the present study are Total ash content, Acid insoluble ash content, Water soluble ash content, Ethanol soluble extractives, Water soluble extractives and Moisture content. The procedures followed for the determination of the various parameters during proximate analysis were according to standard procedures as provided in literature14-15. The moisture content in the plant powders was determined using KarlFischer titration method. Seasonal variation for all the three plants selected for the present work, Aerva lanata, Hedyotis corymbosa and Leptadenia reticulata, have been studied by collecting them in April (summer), August (monsoon) and December (winter) from Vadodara (Gujarat). Geographical variation was studied by collecting these plants from Gujarat, Maharashtra and Kerala. Summer season was selected for collection and only the regions of collection were varied. All the three plants were collected from Vadodara to represent collection from Gujarat. Aerva lanata and Hedyotis corymbosa were collected from Mumbai, while Leptadenia reticulata was collected from Aurangabad. These collections represented collection from Maharashtra. For Kerala collection, Aerva lanata and Hedyotis corymbosa were collected from Allapuzha, while Leptadenia reticulata was collected from Changanacherry. Each sample was analyzed at least three times for confirmation of the results.
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RESULTS
The values calculated for various parameters of the proximate analysis are shown in Table 1

for Aerva lanata, Table 2 for Hedyotis corymbosa and Table 3 for Leptadenia reticulata.

Table 1 Values obtained for the proximate analysis of Aerva lanata


Sr.No Parameter Summer 1. 2. 3. 4. 5. Total Ash Content Acid Insoluble Ash content Water Soluble Ash content Sulphated Ash content Ethanol soluble extractive Water soluble extractive Moisture content 15.480.5 3 2.720.43 8.890.67 20.500.1 3 5.980.14 Gujarat Monsoon 15.840.3 4 3.220.62 8.630.91 22.230.2 4 5.720.87 MeanSD (%)* Maharashtra Winter 16.030.7 3 3.370.58 8.930.45 21.560.4 6 6.120.72 14.110.21 2.980.33 10.780.56 17.890.72 4.290.29

Kerala

9.760.51 0.700.87 5.720.92 14.080.49 8.150.76

6. 7.

13.240.1 8 4.500.83

12.920.6 8 5.130.21

13.160.4 5 4.970.34

10.410.34 4.900.64

18.120.13 5.200.75

*Each value is a mean of 3 readings

Table 2 Values obtained for the proximate analysis of Hedyotis corymbosa


Sr.No. Parameter Summer 1. 2. 3. 4. 5. 6. 7. Total Ash Content Acid Insoluble Ash content Water Soluble Ash content Sulphated Ash content Ethanol soluble extractive Water soluble extractive Moisture content 11.660.63 2.390.59 6.380.34 16.400.34 6.70.12 13.160.20 5.79 0.53 Gujarat Monsoon 11.570.54 2.990.83 6.950.91 15.260.74 6.120.31 12.680.33 5.93 0.43 MeanSD (%)* Maharashtra Winter 11.090.81 2.780.52 6.350.65 15.720.87 6.410.83 12.960.27 5.24 0.19 9.760.64 1.260.76 8.030.53 11.510.81 5.910.62 12.710.40 5.62 0.62 12.230.32 2.470.65 9.290.97 17.850.15 4.650.92 11.710.77 6.96 0.39 Kerala

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Table 3 Values obtained for the proximate analysis of Leptadenia reticulata


Sr.No. Parameter Summer 1. 2. Total Ash Content Acid Insoluble Ash content Water Soluble Ash content Sulphated Ash content Ethanol soluble extractive Water soluble extractive Moisture content Gujarat Monsoon MeanSD (%)* Maharashtra Winter 9.050.86 0.840.43 10.760.72 0.910.78

Kerala

11.480.45 10.180.51 1.290.42 0.970.75

8.760.65 0.940.32

3.

7.070.59

5.30.29

4.920.36

6.090.57

4.170.30

4.

16.880.73 13.660.43 12.240.77

13.690.84

11.210.72

5.

6.280.21

5.970.57

6.590.75

5.130.63

7.920.81

6.

14.640.31 14.010.24 14.310.39

13.890.29

16.120.69

7.

5.330.87

7.28 0.39

5.16 0.51

5.710.19

8.690.48

*Each value is a mean of 3 readings

DISCUSSION
The Total Ash content mainly is a measure of the presence of inorganic compounds in a drug. A larger value indicates that the plant material contains more of inorganic compounds. Concentrated acid, when added to ash, reacts with the calcium oxalate crystals. If the plant material contains a large number of calcium oxalate crystals, the amount of substance remaining after acid treatment will be quite less. Thus a lower value of Acid insoluble ash content indicates the presence of large number of calcium oxalate crystals in the plant material, and the vice versa. Water soluble ash content gives the crude estimate of the water soluble extractable matter present in the ash. Sulphated ash content gives an estimate of the lignin content that remains after treatment with concentrated sulphuric acid. Most animal studies and clinical trials make use of aqueous and ethyl alcoholic extracts. The amount of matter extracted from the plant when water or
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ethyl alcohol is used as extracting solvents is determined by the values of the Water Soluble and Ethanol Soluble Extractives. Moisture content in samples is another parameter that is important to be studied. In a given amount of sample, how much percentage corresponds to that of the water content and how much corresponds to the phytochemicals that are actually active in the plant, is important to be analyzed to decide the correct dosage of the plant material to be administered. Proximate analysis of Aerva lanata: The Total ash content for the plant showed similar and comparable values among the samples collected during three different seasons, indicating consistency in the ash content, irrespective of the season of collection. The sample from Maharashtra was quite comparable to that of the Gujarat sample, while that of the Kerala sample was found to very less as compared to the other two
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samples. Lower value of Acid Insoluble Ash in Kerala sample indicated higher content of calcium oxalate. Other two samples from Maharashtra and Gujarat samples showed larger values, indicating lower calcium oxalate content in the samples collected from both samples. Kerala sample showed the lowest amount of Water soluble ash content as compared to the samples collected from the other two places. The three different seasons showed similar values indicating that the values did not change for the plant from season to season. Gujarat samples collected in all the three seasons showed large values of Sulphated ash as compared to Maharashtra and Kerala samples which showed lower values. Water and ethanol soluble extractive values were highest in the Kerala samples, indicating the richness of the plant material with regard to its phytochemicals. Both the extractive values were least in case of Maharashtra sample. The extractive values of the samples collected from Gujarat collected in three different seasons also showed values that indicated the presence of a good amount of phytochemical content. Moisture content of the plant collected during monsoon season was observed to be greater than those collected during summers and winters, which was expected since the plant has better access to water during the rains. Since water is available in plenty in Kerala, as compared to Maharashtra and the arid Gujarat, the moisture content was found to be the greatest in the Kerala sample as compared to those collected from the other two places. Proximate analysis of Hedyotis corymbosa: With regard to different seasons the Total ash content for the plant did not show much variation, showing similar and comparable values among the three samples, indicating consistency in the ash content. The ash value of the sample from Maharashtra was lesser than that of the Gujarat sample, whereas the sample collected from Kerala showed the highest ash value among all the samples. Hedyotis corymbosa shows the presence of abundant raphide bundles in the leaf as well as the stem. These needle shaped calcium oxalate crystals played an important role in deciding the values of Acid Insoluble Ash in the samples
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collected during the three seasons, as well as from the three regions. Except for the fact that the sample collected from Maharashtra showed a lower value for the same, all other samples showed quite similar values which indicated that the raphide content did not vary from season to season. Kerala sample also showed a value that was comparable to the sample collected from Gujarat. Kerala sample showed the highest amount of Water soluble ash content as compared to the other samples. Gujarat samples collected during three seasons showed the least values, irrespective of the season of collection. Maharashtra sample, however showed an intermediate value, as compared with those of the other two regions. Among the samples collected from Gujarat samples during the three seasons, the summer season showed a large value of Sulphated ash, as compared to the monsoon and winter seasons. The sample collected from Maharashtra showed the least value, and the Kerala sample which showed the largest value among the samples collected from the three different regions. Water and ethanol soluble extractive values were highest in the Gujarat samples, indicating the richness of the plant material in context to its phytochemicals. Both the extractive values were least in case of the sample collected from Kerala. Gujarat samples collected in three different seasons also showed a good amount of extractive phytochemical content, out of which the sample collected during summer season showed the presence of more extractable phytochemicals as compared to the monsoon and winter seasons. Moisture content of the plant collected during monsoon season was observed to be greater than those collected during summers and winters, which was expected since the plant has better access to water during the rains. Since water is available in plenty in Kerala, as compared to Maharashtra and the arid Gujarat, the moisture content was found to be the greatest in the Kerala sample as compared to those collected from the other two places. Proximate analysis of Leptadenia reticulata: The Total ash content for the plant showed a higher value when collected in
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summer, when compared with those collected during monsoon and winter. The plant collected during winter showed the least value of the three seasons. The sample collected from Maharashtra was quite comparable to that of the Gujarat sample, while that of the Kerala sample was found to very less as compared to the other two samples. Lower value of Acid Insoluble Ash in the samples collected from all three different regions indicated higher content of calcium oxalate crystals. Among the plants collected during three different seasons showed the value to be greatest for the summer collection, indicating lower calcium oxalate content for the same. Kerala sample showed the lowest amount of Water soluble ash content, as compared to the samples collected from the other two places. Maharashtra sample showed a higher value. The values when compared for the plant collected in three different seasons indicated that the summer collection indicated highest value, while the winter collection showed the lowest value. Gujarat samples collected in all the three seasons showed large values of Sulphated ash, as compared to Maharashtra and Kerala samples which showed lower values. The Kerala sample showed the least value. The summer collection for the plant showed a higher value, whereas the sample collected during winter showed the least value. Water and ethanol soluble extractive values were highest in the Kerala samples, indicating the richness of the plant material in context to its phytochemicals. Both the extractive values

were least in case of Maharashtra sample. The extractive values of the samples collected from Gujarat collected in three different seasons also showed values that indicated the presence of a good amount of phytochemical content. Moisture content of the plant collected during monsoon season was observed to be greater than those collected during summers and winters, which was expected since the plant has better access to water during the rains. Since water is available in plenty in Kerala, as compared to Maharashtra and the arid Gujarat, the moisture content was found to be the greatest in the Kerala sample as compared to those collected from the other two places. The present study establishes the fact that one cannot assign fixed values for parameters such as ash values, extractive values and moisture content since they are subject to change with variation in season and region of collection. These values however, can help in judging the best time and place of collection for a particular plant. Similar studies need to be carried out for all medicinal plants even if literature quotes values for its proximate analysis. Higher extractive values indicate richness of the plant in terms of phytochemicals. Higher values of sulphated ash indicate greater content of inorganic minerals. Thus a parallel study of a number of samples can help in deciding the best time and place of collection for a particular medicinal plant.

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10. Chang HM and PPH But. Pharmacology and Applications of Chinese Materia Medica. Volume II. World Scientific, Singapore. (1986). 11. Bensky D and A Gamble. Chinese Herbal Medicine: Materia Medica. Eastland Press, Seattle, WA. (1990). 12. Chang Minyi. Anticancer Medicinal Herbs. Human Science and Technology Publishing House, Changsha. (1992).

13. Ou Ming. An Illustrated Guide to Antineoplastic Chinese Herbal Medicine. The Commercial Press, Hong Kong. (1990). 14. The Ayurvedic Pharmacopoeia of India. Volumes I-IV. 1st Edition. NISCAIR, New Delhi. (2004). 15. Quality Control Methods for Medicinal Plant Materials. WHO, Geneva. AITBS Publishers, New Delhi. (2006).

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