Traditional and chemotaxonomic studies of bio guided active constituents of Tinospora cordifolia (Willd.) Hook. F. and Thoms. Root

Introduction

Traditional and complementary medicine (T&CM) is an important and often underestimated health resource with many applications. [1] According to WHO 2002 report, 80% of the world population is still dependent on herbal remedies to treat illness. [2] The Ayurvedic system of medicines is now gaining attention globally due to its efficacy. The standardization of drugs includes Drug authentication, physical parameters, chemical parameters, chromatographic analysis, and microbiological parameters [3] . Pharmaco-therapeutics in the field of Ayurveda, mainly centres on the use of various monoherbal as well as polyherbal formulations for the treatment of various diseases. Specified use of various herbs for numerous ailments can be observed since the Vedic period. Charaka Samhita, one of the two foundational texts of Ayurveda, has enlisted numerous plants, herbs, shrubs and their useful parts that can be used medicinally. The properties of various parts of a single plant can behave differently individually and differently with the combination of some other plant or with some other part of the same or different plant. So as to predict the therapeutic and clinical efficacy of any plant or any part, it is necessary to analyze and evaluate the whole plant according to its chemical properties and its traditional use (if any). Guduchi, botanically known as Tinospora cordifolia Willd Hook Miers, is one of the most widely used herbs in the field of alternative as well as allopathic medicines. It possesses many medicinal and therapeutic properties such as anti-diabetic, antipyretic, antispasmodic, antiinflammatory, anti-arthritic, antioxidant, anti-allergic, anti-stress, anti-leprotic, antimalarial, hepatic-protective, immune-modulatory and anti-neoplastic activities. In ancient and modern Ayurvedic literature Tinospora cordifolia (Guduchi) has been used for the management of diseases like Kamala (hepatitis), Kushtha (skin diseases), Jwara (pyrexia), Prameha (Diabetes and urinary tract disorders), Pandu (anemia)¸Krimi (intestinal worms) and much more. Because of such a wide and effective therapeutic window, T. cordifolia is widely used in various AYUSH formulations Charak Samhita, has also explained the use of Guduchi root as Medhya rasayana (Nootropic enhancing agent). Though the whole plant is of medicinal use stems are leaves are prominently studied and are praised for their therapeutic efficacy in ancient literature also, a large number of studies are available which prove its wide therapeutic efficacy clinically as well as experimentally. Susshrut Samhita, an ancient Ayurvedic Sanskrit text on medicine and surgery, explained a group of herbs called as Valli panchamula, Valli meaning climber, panch means 5 and Mula means root. Thus, Valli panchamula describes a class of five climbers whose root is to be used for medicinal purposes. These 5 plants are grouped together as they possess similar properties and may depict similar chemical composition w.r.t Root. This Valli panchamula, comprises of Vidari (Ipomoea digitata L.), Sariva (Hemidesmus indicus R. Br), Rajani (Rubia cordifolia L.), Guduchi (Tinospora cordifolia Willd Hook Miers) and Ajashrungi (Gymnema sylvestre L.). The group of these 5 plants can be used in the effective treatment of Raktapitta (Haematological disorders), Shopha (Inflammations), Prameha (Urinary disorders and Diabetes mellitus) and Shukradosh (Disorders of semen). Ancient literature of Ayurveda holds the proof of medicinal and therapeutic efficacy of Giloe roots, but conventional research lacks the clinical as well as experimental evidence depicting the use of Guduchi root, thus, here is an attempt to standardize the root of Tinospora cordifolia so that it can be effectively used for the treatment of various diseases.

Materials and Methodology

Experimental analysis can be helpful in assuring the quality, purity and authenticity of the given drug. World Health Organization (WHO) stresses the importance of the qualitative and quantitative methods for characterizing the samples, quantification of the biomarkers and/ or chemical markers and the fingerprint profiles [4] . Analytical analysis of the drug including Physics-chemical, Phytochemical, HPTLC, FTIR, and UV-Visible parameters was performed in All India Institute of Ayurveda, Delhi (RRDR laboratory).

Physicochemical study 1. Loss on Drying (LOD)

Around 2 g of air-dried raw drug was kept in a well-ventilated oven for 3 h at 105 ºC. After removing the drug from the oven it was allowed to cool to room temperature by keeping the dried raw drug in a desiccator for 20 min. Then it was again weighed. The loss on drying (moisture content) in percentage was calculated as follows:

Weight of Empty the Petri dish = W empty Weight of the Petri dish with air-dried raw drug = W+ wet Weight of the air-dried raw drug = W + wet -Empty = W wet Weight of the oven-dried (105 ˚C) raw drug = W+ dry -Empty = W dry % LOD =

Extractive values a) In ethanol extract

Around 2.0 g of raw drug was weighed (moisture content was considered). 50 mL of Et OH was added to the raw drug and kept for 24 h at room temperature. After filtering it, 10 mL was taken and evaporated in a water bath. Thereafter, it was heated at 105 ˚C in a well-ventilated oven till constant weight was achieved. The above experiment was performed twice, and an average value was reported. The ethanol extract value was calculated as follows:

Drug weight = (moisture content was subtracted to calculate the drug weight) Empty Beaker weight = empty Beaker +dried Extract weight = w + drug % ETOH extract = %w/ In aqueous extract In a closely related experiment, the water extract value was determined using 50 mL of water instead of ethanol.

Empty Beaker weight = w empty Beaker + dried Extract weight = w +drug % H 2 O extract = %w/w 3. Ash values a. Determination of total cash value Around 2.0 g of the raw drug was weighed (moisture content was considered) in a thermally resistant previously weighed crucible. The crucible along with its content is heated to 450 ºC for 30 min. The crucible was cooled in a desiccator for 15 min and weighed. Repeated this procedure till constant weight was obtained. Then the percentage of total ash was calculated with reference to the drug weight. Empty Crucible weight= w empty Drug weight= Crucible +ash weight = w + ash Total ash= % Total ash =%w/ w

b. Determination of acid insoluble ash value

The ash obtained (as directed under total cash value) was boiled with 25 ml of 10% HCl for 5 minutes. The insoluble matter was collected on an ash-less filter paper, washed with hot water, and ignited for 15 minutes at a temperature of about 450 °C. The crucible was cooled in a desiccator for 15min and weighed. The heating was repeated till a constant weight was reached. The above experiment was performed twice, and an average value was reported. Empty Crucible weight = w empty Drug weight = Crucible +acid insoluble ash weight = w+ acid (i) ash Acid insoluble ash = w+ acid (i) ashw empty % acid insoluble ash = %w/w

Phytochemical study 1. Preliminary analysis

The study drug was subjected to cold extraction in six solvents namely, 50% hydroalcoholic solution (Ethanol: water), Nhexane, Methanol, Ethanol and Chloroform. 5 gm. of powdered drug was added with 50 ml of each of the solvents. The mixture was then subjected to intermittent stirring for the next 6 hours and then kept stand still for the next 18 hrs. After 24 hrs, the extracts were filtered and filtrates were used for further evaluation of phyto constituents.

A. Estimation of proteins (Million

Analytical analysis 1. HPTLC study

 Standard solution preparation: Standard was prepared in methanol 1:40 mg/ml.  Sample preparation: 1 mg/10 ml sample was prepared.  HPTLC plate images in 254 nm and 366 nm are attached.  From the study, it is found that 4.70 µg of standard compound (Berberine) is present in 100 mg sample.

FTIR study

Fourier transform infrared spectroscopy (FTIR) detects the infrared spectrum of absorption, emission, and photoconductivity of solid, liquid, and gas. Its spectrum is recorded between 4000 and 400 cm -1 [5] Each and every functional group has its own disparate vibrational energy, this energy is used to identify a molecule through the combination of all of the functional groups [12] . The sample absorbs infrared rays at characteristic frequencies depending on its bond strength and structure. The transmitted part of the beam enters the detector for measurement of interferogram signals, which is finally processed by computers providing the absorption data. For analysis of fine powder of Tinospora cordifolia root, the universal attenuated total reflectance (UATR) technique is used, as it is easy and quicker in implementation. FTIR analysis was done using PerkinElmer spectrum. The interpretation of peaks is summed up in Table 07.

UV-Visible analysis

UV-Vis spectroscopy is an analytical technique that measures the number of discrete wavelengths of UV or visible light absorbed or transmitted by a sample compared to a blank or reference sample [7] . This property is influenced by the composition of the sample, which can provide information about what is in the sample and in what concentration. Here, UV spectroscopy of the methanolic extract of Tinospora cordifolia root is performed. The extract was prepared using the cold maceration method in the ratio 1:10 and then subjected to 6 serial dilutions. The extract was analyzed (Analytical Jena specord plus spectrophotometry) and the wavelength of maximum absorption was noted and compared with the wavelength of Berberine. The absorption spectrum is presented in Graph 03. From the analysis, it is evident that maximum UV absorption was obtained at the wavelength 212 nm.

Results and Discussion

References

1. https://eijppr.com/storage/models/article/2nwf6uc0hahi9hi3 xpt9dbpxeh1nqyj2lsvzsgpfq6rdwx6mabswf31hixod/importa nce-and-scope-of-standardization-of-drugs-in-indianmedicine.pdf (accessed on 22/05/2022)
2. Dr. Kiran Kumar Akka, Dr. Pampa Reddy. Lipid profile in patients of type 2 diabetes mellitus with myocardial infarction. International Journal of Advanced Biochemistry Research. 2021;5(1):14-19.
3. Shamsuzzaman MR, Hasan A. Review of anticancer potential medicinal plants. Journal Science Facts. 2019;5:14.
4. Kumar VS, Singh A, Singh AK, Dixit B, Srivastava GK, Sidhu R, et al. Phytochemical, antioxidant, antimicrobial, and protein binding qualities of hydro-ethanolic extract of Tinospora cordifolia, JBAPN. 2018;8(3):192-200.
5. Schmitt J, Flemming HC. FTIR-spectroscopy in microbial and material analysis. International Biodeterioration & Biodegradation. 1998;41(1):1-1.