A Comparative phytochemical study of coldwaterextract, hotwater extract and starch form of Tinospora cordifolia (Thunb.) Miers. stems

1.Introduction

Tinospora cordifolia(Thunb.) Miersis a climbing shrub with a succulent stem and leaves measuring 10-20 cm in diameter. Belonging to the Menispermaceae family and is widely distributed in the Indian subcontinent and China. T. cordifolia, known as Guduchi and Amurta in Sanskrit, means “protects the body from diseases” [1]. The roots, stems, and leaves are integral components of the plant, possessing medicinal and nutritional values.T. cordifoliais an important medicinal plant in the Ayurveda system of medicine used in different Ayurveda formulations to treat various ailments such as colds, fever, headache, jaundice, and digestive disorders [2],[3],[4].Different classes of phytochemical compounds, suchas alkaloids, glycosides, aliphatic compounds, diterpenoids, sesquiterpenoids, phenolic compounds, steroids, and polysaccharides, are found in T. cordifolia[5],[6].T. cordifoliais extremely useful in strengthening the immune system and keeping the functions of its various organs in harmony [5],[7].

According to the Ayurveda medicinal system, its Rasa is Tikta (bitter taste), Veerya is Ushna(hot potency) and Vipakais Madhura(post-digestive effect is sweet),Guna includes Laghu and Snigdha (properties are light and oily).It balances Tridosha(three body humors Vata, Pitta and Kapha) in the body [8]. The starch form of T. cordifolia is traditionally used as a household remedy for chronic fever to alleviate it, reduceburning sensation, and increase energy and appetite [9]. In addition to the starch form of T. cordifolia, cold water and hot water extract (decoction) are used in the Traditional and Ayurveda medicinal systems [8],[10]. Therefore, an attempt was made to compare the phytochemical differences of three types of medicinal preparations prepared from T. cordifolia stems using phytochemical screening and Thin Layer Chromatography (TLC) technique.

2.Materials and Methods

2.1. Collectionof Tinospora cordifolia Stems

Stems of T. cordifoliawere collected from home gardens in the Kalutara district between August2023 to October 2023.

2.2. Different medicinal preparations of Tinospora cordifoliastems

2.2.1. Cold water extract:A weight of 60 g of powdered stem was taken, and 480 ml of cold water was added to an earthen pot. The mixture was incubated overnight and filtered through a cotton cloth in the following morning [11].

2.2.2. Hot water decoction:A weight of 60 g of coarsely powdered stem was taken and boiled with 1920 ml of water in an earthen pot over a mild flame till the liquid part was reduced to 240ml[11].

2.2.3. Starch form:The ground coarse powder was taken, and four portions of water were added. The mixture was incubated overnight to settle down. Supernatant water was decanted carefully the following day, without disturbing sediment starch. The starch was washed using a minimum amount of water and kept settling down. The sedimented starch was allowed to air dry[12].

2.3. Phytochemical Screening

The presence or absence of saponins, tannins, phenols, alkaloids, flavonoids, terpenoids, steroids,carbohydrates, proteins and reducing sugars qualitatively detected in cold water extract, hot water extract (decoction) and starch form according to the standard protocols [13], [14].

2.4. Development of ThinLayer Chromatography (TLC)fingerprints

TLC fingerprints were developed for cold water extract, hot water extract (decoction) and starch form of T. cordifolia stems. The starch form of T. cordifoliawas extracted into Dichloromethane. Extracts were concentrated separately using Rotavapor (Buchi, B- 480). A volume of 10µl from each extract was applied as separate spots on TLC plates. Cyclohexane, Dichloromethane, Ethyl acetate and Methanol were used in a ratio of 6: 4: 1: 0.1 (v/v) as the mobile phase. The TLC fingerprints visualized under UV light (at 254 nm and 366 nm). Subsequently, Vanillin-sulphuric acid was sprayed and heated at 100-110 ^oC. Finally, R_f values were recorded for each spot before (at 254 nm and 366 nm) and after spraying Vanillin-sulphuric acid.

3. Results and Discussion

Different phytochemical classes such as tannins, alkaloids, phenolic compounds, flavonoids, alkaloids, terpenoids, steroids, saponins, carbohydrates, proteins, and reducing sugars present or absent in the cold water extract, hot water extract (decoction) and starch form of T. cordifolia stem are listed in Table 1. 

+:Positive response++:Intensely positive response          – :Negativeresponse

Results revealed that among the tested phytochemical classes, terpenoids and steroids were absent in the cold-water extract, hot water extract (decoction) and starch form prepared from stems of T. cordifoliagrown in Sri Lanka.However, terpenoids such as tinosporide, furano-lactone diterpene, furanolactone clerodane diterpene, furanoid diterpene and steroids such as giloinsterol, ß-gitosterol, 20a- hydroxy ecdysone were reported to be presented in T. cordifoliagrown in other countries [15]. Moreover, alkaloids, saponins and carbohydrates were prominent in the starch form of T. cordifolia stem.

Thin Layer Chromatography (TLC) is a rapid, inexpensive, and simple technique used to identify active ingredients, determine the identity of the medicinal plants or polyherbal preparations and detect adulterants, contamination or substitution [16],[17],[18]. In the present study, TLC fingerprint was examined under UV light at wavelengths of 254 nm and 366 nm and sprayed Vanillin-sulphuric acid to visualize the colors (Figure 1). Moreover, R_f values were recorded forcold water extract, hot water extract (decoction) and starch form prepared from T. cordifolia stems (Table 2).

As per the TLC fingerprint profiles (Figure 1) and their R_f values, both cold water extract and hot water extract (decoction) have similarities whereas the starch form of T. cordifoliadiffers from cold water extract and hot water extract (decoction). In conclusion, the presence study revealed the phytochemical differences of cold water extract, hot water extract (decoction) and starch form of T. cordifolia stem for the first time.

Competing interests:

The authors declare no competing interests.

References

1.George, M., Joseph, L., Mathew, M. 2016. Tinospora cordifolia; A pharmacognostical update. The Pharm Innov. 5(7): 108 – 111.

2. Prajwala, B., Raghu, N., Gopenath, T.S., Basa lingappa, K.M. 2019. Guduchi: Its medicinal properties. J Plant PhysiolPathol. 7:3.

3. Saxena, C., Rawat,G. 2019. Tinospora cordifolia (Giloy) – therapeutic uses and importance: a review.Curr Res Pharm Sci. 9 (3):42-45.

4. Valte, V.S., Atlarde, D. 2022. A brief review on Guduchi (Tinospora cordifolia). Int J Pharm Sci Res. 13(5):1818 – 1832.

5. Akram, M., Hamid, A.,Khalil, A.,Ghaffar, A.,Tayyaba, N.,Saeed, A et al.Review on medicinal uses, pharmacological, phytochemistry and immunomodulatory activity of plants. Int J ImmunopatholPharmacol. 27 (3):313-319.

6. Ahmad, W.,Jantan, I.,Bukhari, S.N.A. 2016.Tinosporacrispa (L.) Hook. f. & Thomson: a review of its ethnobotanical, phytochemical, and pharmacological aspects. Front Pharmacol. 7:1-19.

7. Manjrekar, P.N., Jolly, C.I., Narayanan, S. 2000. Comparative studies of the immunomodulatory activity of Tinospora cordifolia and Tinospora sinensis. Fitoterapia. 71:254–7.

8. The Ayurvedic Pharmacopoeia of India.2001. Part I. 1st ed. Vol. 1. New Delhi: Department Of AYUSH, Ministry of Health and FW. 53–5.

9.Sharma, R., Amin, H., Galib, R., Prajapati, P.K.2013. Seasonal variations in physicochemical profiles of GuduchiSatva (starchy substance from Tinospora cordifolia [Willd.] Miers). J Ayurveda Integr Med.4(4):193–197.

10. Upadhyay, A.K., Tinospora cordifolia (Willd.) Hook. f. and Thoms. (Guduchi) – validation of the Ayurvedic pharmacology through experimental and clinical studies. Int J Ayurveda Res. 2010 Apr-Jun;1(2):112–121.

11. Bramhanand Tripathi, 2016.Sharandhar Samhita (Madhyam Khanda), ChaukhambaSurbhartiPrakashan, Varanasi, Edition.

12. Chunekar, K.C.,Bhavaprakash, Nighantu. 2013. Varanasi Chaukhamba Bharati Academy:257, Hariprapanna Sharma Rasa Yoga Sagara. Varanasi ChaukhambaKrishnadas Academy.

13. Hewageegana, S., Arawwawala, M., Fernando, P, Dhammarathana, I., Ariyawansa, S. 2014. A comparative study of physico-chemical and phytochemical parameters: glands/hairs of the fruits and the leaves of Mallotusphilipinensis (Lam.) Muell. Arg.  grown in Sri Lanka. J Natl Sci Found. 42: 291 -295.

14. Karunakaran, R., Thabrew, M.I., Thammitiyagodage, G.M., Galhena, B.P., Arawwawala, L.D.A.M. 2017. The gastroprotective effect of ethyl acetate fraction of hot water extract of Trichosanthescucumerina Linn and its underlying mechanisms. BMC Complement Altern Med. 17:312.

15.Sharma, P., Dwivedee, B.P., Bisht, D., Dash, A.K., Kumar, D. 2019.The chemical constituents and diverse pharmacological importance of Tinospora cordifolia. Heliyon. 12,5(9):e02437.

16. Arambewela, L.S.R., Arawwawala, L.D.AM. 2010. Standardization of Alpinia calcarata Roscoe rhizomes. Pharmacognosy Res. 2(5):285–288.

17.Karunaratne, T.D.N., Sugataratana, K., Ariyawansa, H.A.S., Silva, H.A.D., Samarasingha, K., Arawwawala, L.D.A.M. 2015. Standardization of SarasvathaChoorna: Used as a remedy for dementia. American J ClinExp Med. 3: 288-292.

18. Hewageegana H.G.S.P., Arawwawala L.D.A.M. 2018. Development of Thin Layer fingerprint profiles for Leptadenia reticulate (Rez) Wight and Arn and its substitutes. Int J Ayurveda. 3(5): 1-4.