Phytochemical and Antimicrobial Activity of Fumes and Powder Extracts of Tinospora cordifolia

. Herbal medicines have a long therapeutic history serving large population of the world. However, the quality con-trol and quality assurance still remains a challenge because of the high variability of chemical components. Tinospora cordi-folia is commonly referred to as Amrita or Giloy or Guduchi . All the parts of the plant are immensely useful due to the presence of different secondary metabolites. We aimed to investigate the qualitative and quantitative (carbohydrate and poly-phenolic compound) analysis of secondary metabolites present in extracts of T. cordifolia powder and its herbal fume (generated from Yagya), followed by the study of its antimicrobial activity against E. coli and P. aeruginosa . The present study’s powder and fume cocktail (hexane, ethyl-ether and methanol) extracts (10 mg/ml) showed, respectively 30 mm and 21.67 mm Zone of inhibition against E.coli indicating equivalent eﬀiciency of herbal fumes. Similarly, fumes contained all secondary metabolites (qualitative testing) similar to that of powder extract. In addition, carbohydrate and poly-phenolic compound present in the fume extracts (0.277 mg/g and 0.29mg/g), were equivalent or higher than powder extracts (0.131 mg/g and 0.26mg/g) indicating holistic utility of fumes of T. cordifolia


Introduction
Herbal medicines have a long therapeutic history and are still serving many of the health needs of a large population of the world. However, quality control and quality assurance still remain a challenge because of the high variability of chemical components involved. The genus Tinospora (Menispermaceae), is a perennial, deciduous, large, glabrous, climbing shrub with a weak and fleshy stem, found throughout Asia, Africa, and Australia. The prominent member of the Tinospora genus, out of 34 different species, is T. cordifolia, which is a well-recognized and widely distributed traditional plant that is used successfully in Indian Ayurveda medicine and is found throughout the tropical Indian subcontinent and China [1]. It has numerous ethnomedicinal applications in Ayurvedic pharmacology. The plant T. cordifolia is a stem and in Ayurvedic practice, generally a decoction of Amrita is given to the patient.
All the parts of the plant are immensely useful due to the presence of different secondary metabolites of pharmaceutical importance belonging to various groups such as steroids, diterpenoid lactones, sesquiterpenoids, glycosides, alkaloids, and phenolics [2]. It was reported that Guduchi also possesses adaptogenic potential. The decoction is prepared alone or in combination with other herbs [3]. According to the Bower Manuscript a medical treatise, recorded in the 6 th century A.D. in the Sanskrit language on the birch bark leaves, T. cordifolia is used in the recipe for the preparation for 'Amrita-Prâsa clarified butter' and 'Amrita Oil', which is considered as soma (food for immortality) [4]. T. cordifolia has shown many promising biological activities, such as anti-oxidative, antimicrobial, antihyperglycemic, anti-inflammatory, osteoprotective, hepatoprotective, antidiarrheal, and antistress effects. T. cordifolia is a rich source of protein as well as micro-nutrients such as iron, zinc, copper, calcium, phosphorus, and manganese. Terpenes, alkaloids, flavonoids, steroids, and glycosides are among the secondary plant metabolites found in it [5]. The genus Tinospora is reported to contain over two hundred different phytochemicals from diverse chemical classes in phytochemical characterization, with diterpenoids representing the most abundant chemical class [6]. We aimed to investigate the differences between the qualitative and few quantitative analysis of secondary metabolites present in extracts of T. cordifolia powder and its herbal fume (generated from Yagya), followed by the study of its antimicrobial activity against E. coli and P. aeruginosa.

Sample collection
Dried Sample of T. cordifolia was collected from the Shantikunj Pharmacy, Dev Sanskriti University, Shantikunj-Haridwar, India.

Herbal fume preparation via the process of Yagya
The Yagya was performed using mango wood (400gm), ghee (clarified butter) (40 gm) and an herbal mixture of dried T. cordifolia stem powder in a Yagya-chamber (12.5 X 12.5 feet room) with closed controlled conditions. The herbal fumes were collected with the help of an air sampler (APM 433) from Envirotech Instruments Pvt. Ltd, Delhi.The fumes generated by Yagya were subjected to an air sampler present inside the Yagya chamber. The herbal fumes were absorbed in air sampler impingers containing solvent system of hexane, ethyle-ether and methanol in a 1:1:1 ratio. The resultant solution of impinger was subjected to a rotary evaporator at 60℃ and when about 1 ml of sample was been left, it was further subjected to a freeze drying at -40℃. The resultant power of herbal fumes was prepared using DMSO (Dimethyl Sulfoxide) and methanol in various dose concentrations. The herbal fumes preserved in DMSO were subjected to antimicrobial analysis while in methanol were used in phytochemical analysis.

Herbal powder extraction of T. cordifolia
Powdered stem materials (5 gram) were extracted in soxhlet with 20 ml of a 1:1:1 solvent system of hexane, ether, and methanol. The extract was filtered and then the filtrate was subjected to a rotary evaporator at 60℃ and when about 5 ml of sample was left, it was further subjected to a lyophilizer. These extracts were re-suspended in DMSO for further analysis.

Chemical characterization of herbal fumes and herbal powder
Qualitative and quantitative chemical characterization of the herbal fumes and herbal powder was performed [8,9]. The secondary metabolites analyzed for qualitative analysis were carbohydrate, poly-phenol, flavonoid, alkaloids, tannins, and for quantitative analysis were polyphenol [10] and carbohydrate [8,9].

Antimicrobial action of herbal fumes
The antimicrobial activity of herbal fumes and herbal powder was analysed by the kirbybauer agar well diffusion method [11] against E. coli and P. aeruginosa. Inoculums were prepared by direct colony suspension method. A small volume of sterile water was poured inside a test tube to which 50 µl of 24 hr broth culture was added until it matched the 0.5 McFarland standard turbidity. The dose concentration used for antimicrobial analysis was 10 mg/ml.

Name of bacteria
Zone of inhibition (in mm)

Results and Discussion
There are ample descriptions of Yagya in Vedic literature that refer to its multiple applications such as social development, spiritual development, ecological balance, and preventing and curing diseases. Among them, one of the most important aspect is health applications.
The therapeutic application of Yagya is known as Yagya Therapy or Yagyopathy. It has been observed that Vedic literature describes the treatment of a wide range of diseases (both infectious and non-infectious) and ailments through Yagya, along with the precautions and procedures [12].   [13]. T. cordifolia calms the mucous membrane of the respiratory system and hence its herbal fume can be used to treat inflammation due to its anti-inflammatory and antioxidant properties [14].
The Yagya is a holistic treatment. In Ayurveda, the application of medicinal-fume for therapeutic purposes has been discussed in detail. Charak Samhita 5/25-61 describes the use of herbal fume inhalation as a technique to cure or prevent diseases, which is similar to Yagya fume inhalation for treatment [15]. In the present study, dried stems of T. cordifolia is used for phytochemical analysis and antimicrobial activity against bacteria E. coli and P. aeruginosa responsible for multiple diseases like lung disorder, skin disease, colon infection, and pneumonia, and it has been found that 10 mg/ml T. cordifolia of have antimicrobial activity against E.coli and P. aeruginosa with the zone of inhibition of 21.67 mm and 25 mm in case of herbal fumes obtained by Yagya, while in herbal powder of T. cordifolia the zone of inhibition observed is 30 mm in E. coli and 23.5 mm in P. aeruginosa ( Table 1). The herbal powder had more antimicrobial activity against E.coli while in the case of P. aeruginosa the results were nearly the same (Table 1). Further study quantifying the comparison between fume and powder is required.

Phytochemical Analysis of Tinospora cordifolia
The ethnomedicine literature, which includes traditional ancient Indian scriptures and Ayurvedic medicine, is rich in pre-clinical and clinical substantiation data that supports the immunomodulatory-related structures and functions of T. cordifolia herbal preparations. The present study speaks about T. cordifolia as a rich source of antioxidant components both in powder and fume forms.
Phenolic compounds act as antioxidant agents by scavenging the free radicals due to the presence of hydroxyl groups in them [16,17], and they also act as reducing agents, hydrogen donors, metal chelators and singlet oxygen quenchers due to their redox properties [18]. Phenolic acids and flavonoids are considered typical phenolic compounds which possess antioxidant activity and they are widely distributed in the plant kingdom [19]. The use of synthetic antioxidants has been restricted due to their health risks and toxicity. The present study, it was found that the poly phenolic compound present in herbal powder of the T. cordifolia is 0.26 mg/g and in herbal fumes generated by Yagya is 0.29 mg/g (Supplemental Figure 1). The total carbohydrate content in the herbal fumes of Yagya is 0.277 mg/g, which is 53% more than the carbohydrate content found in herbal powder, which is 0.131 mg/g (Supplemental Figure 1), which might be due to mango wood fume along with herbal mixture fume. Also the herbal fume activity compared to its powder extract was less against E. coli, which is gut bacteria, suggesting holistic benefits of herbal fume of T. cordifolia and Mango. This result reflected the ancient practice of Yagya for building strength and energy in the body.
The qualitative analysis of phytochemicals of herbal fumes and herbal powder was also studied (Table 2). In the fumes, flavonoids, alkaloids, tannins, proteins, cardiac glycosides and carbohydrates were present, while in herbal powder cardiac glycosides, tannins, and alkaloids were absent, while others were present ( Table 2). The study conducted by Garg et al. suggested that cardiac-glycosides, alkaloid, and carbohydrates were absent in the herbal powder of T. cordifolia (stems) [20], which were similar to the current findings; the only difference was that carbohydrates were present in our findings. The extract of herbal fumes generated by Yagya contained all of these secondary metabolites indicating its holistic utility.
In another study performed by Jeyachandran et al. it was found that T. cordifolia dried stems, aqueous, ethanol and chloroform extract of 100 mg/ml had antimicrobial activity against E. coli with 4 mm, 9 mm and 8 mm zones of inhibition, respectively, while in the present study, the cocktail extract ( hexane, ethyle-ether and methanol in 1:1:1 ratio) showed a 30 mm zone of inhibition against E. coli [21], which was very higher than ethanolic extract alone; the fume also had higher zone of inhibition indicating efficient extract of all secondary metabolites and hence its holistic utility. The herbal fume extract also had similar extraction efficiency to that of powder extract in terms of poly-phenolic content. Prasad and Chouhan studied the antimicrobial and antioxidant activity of T. cordifolia. The authors reported that the highest total poly-phenolic content was 0.31 mg/g of methanol, which was close to the present findings, which was 0.29 mg/g in herbal fumes and 0.26 mg/g in herbal powder of fumes [22].
As the incidence and susceptibility to acute and chronic diseases continuously increase, many health-conscious individuals have shifted their mindset to a holistic treatment that incorporates measures and routines that promote disease prevention and resilience. The process of Yagya, which is offering medicated herbal mixture to fire, facilitates the aim of holistic treatment of the person. The ancient Indian practices were well aware of the nutrient level, phytochemical utility, and antimicrobial application of Guduchi (T. cordifolia) and used this herb in Yagya.
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Conflict of interest
The authors declare that they have no conflict of interest.