INTRODUCTION:

The drug Soma is considered as a divine medicinal plant with multidisciplinary properties. As the name indicates it is a divine and sacred plant with miraculous properties. Information about Somalata is found in all the Vedic literature such as Rig-veda, Yajur-veda, Sam-veda, Atharva-veda also in Brahmanas and Samhitas. The botanical identity of the drug Soma is still being controversy since different plants are being used under the name Soma in different regions of India.

The major plants used as Somalata are Cynanchum viminale (Sarcostemma acidum Roxb.), Sarcostemma brevistigma W & A; Sarcostemma viminale (L.) R.Br; Asclepias acida (Roxb.), Ceropegia juncea (Roxb.), Ruta graveolens and Ephedra sp. As per a previous report Cynanchum viminale and Ceropegia juncea were grouped as substitutes as they yield alcohol on fermentation and their similarities in nature^1,2.

Both C. viminale and C. juncea belong to the family Apocynaceae. Whole plant of C. viminale is being used in traditional systems of medicines and is found in countries like Burma, China, Nepal, Thailand, Vietnam and India. The plant is considered emetic, alternative, narcotic and rejuvenating and used in hydrophobia and psychopathy. It is reported to possess anti- rheumatic, anti-allergic, antiemetic and bronchodilatory activities.¹ Ethyl acetate extract of C. viminale showed remarkable anti-inflammatory activity and good anti-psychotic efficiency. Ethyl acetate extract is effective on Central Nervous System inhibitory property also. Various lignans³, phenolics compounds⁴and flavonoids^5,6 were reported from this plant earlier.

C. juncea is found mainly in Western Ghats of India especially in Kerala, Karnataka, Andhra Pradesh, Tamil Nadu, Maharashtra and also in Sri Lanka. It is reported to possess antipyretic, analgesic, local anesthetic, antiulcer, hepatoprotective and hypotensive activities¹.Rare Alkaloids were reported from this plant earlier. The plant also showed. The alkaloidal fraction exhibited promising mast-cell stabilizing, tranquilising and hypotensive activities and was devoid of side effects^7,8.

Comparative phytochemical analysis of various source plants used for an Ayurvedic drug is important as it exposes different phytochemical entities present in them. The plants selected such as C. viminale and C. juncea were using as official source plants in traditional Indian medicine. In the present study High performance thin layer chromatography method was applied as the major tool for comparative analysis⁹. It has wide range of applications in modern science. This is being used as an effective tool in analyzing and quantifying the individual components in modern pharmaceutical medicines^10-13. HPTLC is the most simple and efficient tool in analyzing the chemical compounds present in and herbal medicine^14,15. It has been using in standardizing formulations in ayurveda^17,18. The present study was focused on the detailed chromatographic analyses such as high-performance thin layer chromatography and high-pressure liquid chromatography as tools to distinguish the similarities and dissimilarities of chemical nature of the two plants. To the best of our knowledge this is the first attempt to compare phytochemically the two source plants of Soma.

MATERIALS AND METHODS:

Collection of plant materials:

The source plants of Soma such as C. viminale and C. juncea were collected from the nursery of Centre for Medicinal Plants Research, Arya Vaidya Sala, Kottakkal, Kerala, India.

Chemicals used:

All solvents and chemicals were of analytical grade and purchased from Merck, India. Standard Lupeol and Stigmasterol were purchased from sigma Aldrich Bangalore.

Preparation of extracts:

The plant materials were shade dried and pulverized. 3 gram each of the sample was extracted with solvents like n-hexane, chloroform and methanol, ethanol and water using solvent extractor (VELP Scientifica, Italy), with one hour immersion followed by 30 minutes washing and solvents were recovered in 30 minutes. The final extracts were concentrated using rotary evaporator (Heidolph, Germany) and made up to 50ml with respective solvents in standard flasks. The extracts were kept under refrigerator until the various phytochemical analyses.

Preliminary phytochemical screening:

The ethanolic extract of both plants were subjected to preliminary phytochemical analysis using standard procedures for the qualitative detection of major class of phytochemicals¹⁹.

Estimation of total phenolic content (TPC):

The total phenolic content was determined by using the Folin-Ciocalteu assay. An aliquot (1ml) of extracts or standard solution of Gallic acid (20, 40, 60, 80 and 100 µg/ml) was added to 25ml of volumetric flask, containing 9ml of distilled water. 1ml of Folin-Ciocalteu phenol reagent and after 5 minutes 10ml of 7% Na₂CO₃ solution was added to the mixture. After incubation for 90 minutes at room temperature, the absorbance against reagent blank was determined at 550nm with a UV-Visible spectrometer²⁰.

Estimation of total flavonoid content (TFC):

Total flavonoid content was measured by the aluminium chloride colorimetric assay. An aliquot (1ml) of extracts or standard solutions of quercetin (20, 40, 60, 80 and 100µg/ml) was added to volumetric flask containing 4 ml of distilled water. Add 0.30ml 5% NaNO₂ to the flask was and after five minutes 0.3ml 10 % AlCl_3. After five minutes, 2ml 1M NaOH was added and the volume was made up to 10ml with distilled water. The solution was mixed and absorbance was measured against blank at 510nm²¹.

Estimation of total carbohydrate:

Carbohydrate content in the samples was estimated by anthrone method. 100mg of the samples was hydrolysed by keeping on water bath for 2 hours with 5ml of 2.5N HCl and cooled to room temperature. Neutralize it with Sodium carbonate until the effervescence ceases. Made up the volume to 100ml, from this 0.5 and 1ml aliquots were used for analysis. Standard glucose solutions (0.2, 0.4, 0.8 and 1ml) and a blank were prepared. To 1ml of sample 4ml of anthrone reagent was added. The tubes were heated in a boiling water bath and cooled rapidly. Green colour thus formed was read at 630nm. Standard graph were drawn by plotting concentration on the X-axis versus absorbance on the Y-axis. From the graph the amount of carbohydrate present in the samples was calculated²².

Estimation of total free amino acid:

500mg of the plant material extracted with 10ml of 80% ethanol and suspension was filtered and the marc was re-extracted thrice. The filtrates were combined and reduced the volume to 5ml under vacuum. To 0.1ml of extract, 1ml of ninhydrine solution was added and the volume was made up to 2ml with distilled water. Standard solutions of L-alanine (containing 10, 20, 30 40 and 50mg/ml of) were prepared in the similar manner and a reagent blank. All the tubes were kept in water bath and after cooling 5ml of n-propanol: water (1:1) was added and mixed gently. After15 min, intensity of the purple colour was measured against the reagent in a colorimeter at 570nm²³.

High performance thin layer chromatography:

HPTLC analyses were performed on aluminium backed pre-coated silica gel 60 F₂₅₄ TLC plates. Samples were applied to the plates by means of CAMAG Automatic sampler V. Development of plates was done using respective solvent systems in TLC chamber to a distance of 9cm.TLC images were documented using CAMAG TLC visualizer under 254nm, 366nm and in visible light after derivatizing with anisaldehyde sulphuric acid reagent. Desitometric scanning of the developed plates was performed with a CAMAG TLC Scanner. Integrated Software Wincats was used for the evaluation of data. The R_f values of each separated bands were noted.10µl of each of sample solutions were applied on TLC plate. Solvent systems used for different extracts were seq. n-hexane - toluene: ethyl acetate (8:2), seq. chloroform - toluene: ethyl acetate (7:3), seq. methanol - toluene: ethyl acetate (6:4), crude ethanol - toluene: ethyl acetate: methanol (5:5:0.5), crude water- ethyl acetate: methanol: acetic acid: formic acid (9:2:1:1).

Identification of Lupeol and Stigmasterol:

10ml of ethanolic extracts of C. viminale, C. juncea and 3ml of lupeol and stigmasterol were spotted on TLC plate. The plates were developed using the solvent system toluene and ethyl acetate (7:3) in TLC chamber to a distance of 9cm. The plates were derivatised using ANS reagent followed by heating at 105⁰C for 5 min and photographs were recorded. Desitometric scanning was performed by using TLC Scanner 3 at 550nm.

High pressure liquid chromatography:

HPLC analysis was carried out using Shimadzu High Performance Liquid Chromatographic system equipped with LC-10ATVP pump, SPD M10AVP Photo Diode Array Detector in combination with CLASS-VP 6.12 SP5 integration software. Gradient elution was performed with methanol (solvent A) and water (solvent B) in a binary gradient flow by changing the concentration of solvent B; 0-5 min 70%; 5-10 min 60%; 10-15 min 50%. 15-20; 40%, 20-25 min, 30%; 25-30 min, 50%. The DAD signal was recorded at 360 nm.

RESULTS:

Preliminary phytochemical screening:

Major phytochemical groups such as alkaloids, phenolic compounds, flavonoids, phytosterols, saponins, carbohydrates, proteins and amino acids were found in ethanolic extracts of both plants. Glycosides were found absent in both extracts. The contents of phytochemical were differing in their percentage in both plants. The quantity of alkaloids was found higher in C. juncea compared to C. viminale. Saponin content was also found to be higher in C. juncea.

Estimation of total phenolic and flavonoid content:

Total phenolic content was expressed as milligram equivalent of gallic acid (mg GaE). In both C. viminale and C. juncea phenolic content was found almost equal with a difference of 0.4 mg GaE. The phenolic estimation reveled that both species contain moderate quantity of phenolic compounds.

Total flavonoid content was expressed as milligram equivalent of quercetin (mg QE) per gram of extract. C. viminale observed highest flavonoid content and their difference in percentage was 0.2mg QE/g. The values of flavonoid content shows that major phenolic type of compound in both plants extracts were flavonoids.

Estimation of carbohydrate and total free amino acid:

The total carbohydrate content was calculated on the basis of calibration curve of standard glucose. The carbohydrate content was found higher in C. juncea. than C. viminale with a difference of 0.7mg/g. The total free amino acid content was estimated based on the calibration curve of standard L-alanine. The amount of total free amino acid was found high in C. juncea when compared with C. viminale. Quantitative estimations of major group of compounds in two plants were found almost comparable percentages.

Table 1. Results of various quantitative assays of C. viminale and C. juncea

Total phenolic content

(mg GaE/g)

Total flavonoid content

(mg QE/g)

Total carbohydrate content (mg/g)

Total amino acid content (mg/g)

C. viminale

6.3±0.5

5.4±0.1

2.1±0.4

21.6±0.5

C. juncea

6.7±0.3

5.2±0.3

2.8±0.5

38 ±0.3

High-performance thin layer chromatography analysis:

HPTLC profiles of hexane extracts of plants on evaluating under 254nm, showed similar chemical quenching bands at 0.13, 0.66 and 0.81. Under 366nm, both plants show major bands at 0.28, 0.66 and 0.83 with red fluorescence. After derivatizing, they showed green colored band at 0.38 and blue colour bands at 0.55 and 0.71. Chloroform extracts under 254nm, showed chemical quenching at 0.32, 0.76 and 0.87. On evaluating under 366nm, they showed major bands at 0.04, 0.30, and 0.76 with red fluorescence. After derivatizing with ANS, both showed blue colored bands at 0.17 and 0.61, green color band at 0.85. The HPTLC profiles of hexane and chloroform extracts were found almost similar or chemical compounds were found common in both.

Figure 1. HPTLC profile of hexane extracts of C. viminale and C. juncea

Figure 2. HPTLC profile of chloroform extracts of C. viminale and C. juncea

Methanolic extracts under 254 nm showed slight different chemical quenching pattern. At 366 nm both showed major bands at 0.06, 0.46 and 0.76 with red fluorescence. But the concentrations of bands were higher in C. viminale. After derivatization the profiles were almost similar. Crude ethanolic extracts of plants under 254 nm, showed similar bands at R_f 0.11, 0.26, 0.63 and 0.78. At 366 nm, both plants showed similar bands at 0.27, 0.45 and 0.51, but the percentage of compounds was found higher in C. viminale. After derivatization with ANS reagent C. viminale showed more compounds. Methanolic and ethanolic extracts of both plants also showed almost related chemical profiles with some differences. HPTLC profiling of aqueous extracts of both plants showed some difference in some bands at 254 nm and 366 nm. After derivatisation with ANS reagent C. viminale and C. juncea showed almost similar banding pattern and profiles were almost comparable.

Figure 3. HPTLC profile of methanol extracts of C. viminale and C. juncea

Figure 4. HPTLC profile of ethanol extracts of C. viminale and C. juncea

Figure 5. HPTLC profile of water extracts of C. viminale and C. juncea

The HPTLC profiling for the identification of lupeol in C. viminale and C. juncea was carried out using suitable solvent system and after derivatizing with ANS regent both showed similar violet colour band at R_f0.81similar with standard lupeol. The HPTLC densitometric scanning profiles showed that area percentage of lupeol in C. viminale was higher than C. juncea.

HPTLC profiling for stigmasterol identification was done using suitable solvent system. And after derivetizing with ANS reagent the ethanolic extracts of both plants showed similar blue colour band at R_f 0.68 similar to standard stigmasterol. The HPTLC densitometric scanning profiles showed that area percentage of stigmasterol in C. viminale was higher than C. juncea.

Figure 6. HPTLC identification of stigmasterol and lupeol in C. viminale and C. juncea

1. C. viminale; 2. C. juncea; 3. Stigmasterol; 4. Lupeol

Figure 7. HPLC chromatograms of C. viminale and C. juncea

High pressure liquid chromatography analysis:

High pressure liquid chromatography analysis of the ethanolic extracts of both plants was done in same solvent system. The profiles were almost similar at 360 nm. Peaks at R_t 1.7, 3.3, 3.6 and 4.3 were observed in both plants. Peaks at 21.0, 24.5 and 30.9 were observed in high percentage in C. viminale than C. juncea. Almost all peaks were common in both chromatograms rather than some variations in their concentration. The profiles showed that chemical constituents were almost common in C. viminale and C. juncea.

DISCUSSIONS:

Both the C. viminale and C. juncea contains an average percentage of phenolic compounds and their difference in content was negligible. Phenolic compounds were having important biological properties like antioxidant, anti-cancer, anti-ulcer, anti-microbial and anti-inflammatory etc, have been reported and their structure activity relationship was well established earlier^24,25. Presence of phenolics compounds might have a beneficial effect on the medicinal activities of these plants. Flavonoids content in both plants were also almost similar. The values of flavonoid content shows that major phenolic type of compound in both plants extracts were flavonoids. Flavonoids are highly potent compounds with notable antioxidant and anticancer activities. Various investigations have established a relationship between the structures of different flavonoids and their relative efficiencies as antioxidants.¹⁵ More detailed studies may reveal the nature of phenolic and flavonoid compounds in both plants. Carbohydrates and amino acids were considered as primary metabolites in plants. Estimation of were done for the comparison of their percentages. Carbohydrate percentages in both were almost comparable but the amino acid content showed a variation. C. juncea showed higher amino acid content compared to C. viminale.

High performance thin layer chromatography profiles of hexane, chloroform, methanol, ethanol and water extracts of both C. viminale and C. juncea were developed and analyzed to compare the chemical constituents present. HPTLC profiles of different extracts of C. viminale and C. juncea showed nearly comparable banding pattern, that means the presence of similar chemical compounds. Methanolic and ethanolic extracts of both plants also showed almost related chemical profiles with some differences. Water extract of both showed some similarities and differences. HPTLC is considered as a reliable, effective and economical tool for phytochemical comparison and the study established the identical chemical nature of C. viminale and C. juncea. The extracts of both C. viminale and C. juncea were found rich in terpenoids and sterols compounds. Major terpenoid and sterol compounds present was identified from the extracts using marker compounds. Stigmasterol was identified as the major sterol in both plants and which is useful in prevention of cancers, lowering serum cholesterol level and used as a precursor of synthetic progesterone and precursor of vitamin D3²⁶. Lupeol was identified as the major terpenoid in both plants, an important bioactive triterpenoid having anti-microbial, anti-inflammatory, anti-tumor, anti-protozoal anti-proliferative, anti-invasive and cholesterol lowering properties²⁷. Characterization of major compounds will help to identify the plants in routine quality control test.

High pressure liquid chromatography analysis of ethanolic extracts of both plants was done in same solvent system for comparison. Almost all peaks were common in both chromatograms rather than some variations in their concentration. The profiles showed that chemical constituents were almost common in C. viminale and C. juncea.

CONCLUSION:

Comparative phytochemical analysis of the source plants used for an Ayurvedic drug is an important concept as it exposes the similarity and differences in phytochemical entities present in them. Plants such as C. viminale and C. juncea used as source plants of Somalata in various regions of the country were compared phytochemically. Phytochemicals such as alkaloids, phenolics, flavonoids and phytosterols etc. were found in both species. However percentages of alkaloids and saponins were higher in C. juncea. C. juncea showed higher contents of phenolics, carbohydrates and amino acids whereas flavonoids were found more in C. viminale. Chromatographic evaluation using HPTLC provided the comparative chemical profiles of two species. On evaluating the R_f values of different bands separated and also on comparing the spectrum scanned in various wavelengths, it is noticed that most of the compounds are common for both species. An active terpenoid, lupeol and a steroid, stigmasterol were identified from both species. HPLC profiles of ethanolic extracts on evaluation showed almost comparable chromatograms. The present study concluded that the phytochemical profiles of C. viminale and C. juncea were almost similar. On the basis of phytoconstituents, these two species can be used as source plants for Soma. The present phytochemical study provides scientific proof for the use of C. viminale and C. juncea as source plant of Soma in Ayurveda.

ACKNOWLEDGEMENTS:

The authors are thankful Dr. Geetha S Pillai and Dr. Satheeshna kumari for providing the plant materials. The financial assistance from Navajbhai Ratan Tata Trust is greatly acknowledged.

DECLARATION OF INTEREST STATEMENT:

No conflicts of interest declare by authors.

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Received on 16.12.2020 Modified on 24.02.2021

Asian J. Pharm. Res. 2021; 11(3):156-162.

DOI: 10.52711/2231-5691.2021.00029