Assessment of Cytotoxic, Antioxidant, Thrombolytic, Anti Inflammatory and Antimicrobial Activity of Curcuma longa Linn, Cissus quadrangularis and Boerhaavia diffusa Herbal Mixture-An In vitro Study

Background and Aim: Plants play an important role in drug research, and the pharmaceutical industry is heavily reliant on natural products for new drug development. Curcumin, a natural compound contained in the rhizomes of the plant Curcuma longa Linn., has been shown to have anti-inflammatory properties in scientific studies. Cissus quadrangularis L. is a fleshy plant that can be found all over the world, especially in Asia, Africa, and a few other warm tropical areas. Boerhaavia diffusa L. (Nyctaginaceae) is present in the tropical regions of India, South America, and Africa. B. diffusa roots are commonly used in Ayurveda to treat various ailments. The aim of Original Research Article Dinesh et al.; JPRI, 33(60B): 1766-1777, 2021; Article no.JPRI.79214 1767 the study is to assess the cytotoxic, antioxidant and thrombolytic, antiinflammatory and antimicrobial properties of the aqua-alcoholic extract of the herbal mixture (Curcuma longa Linn +Cissus quadrangularis +Boerhaavia diffusa ) Methods: Aqua-alcoholic extract of mixture of Curcuma longa Linn, Cissus quadrangularis and Boerhaavia diffusa was prepared. Phytochemical in-vitro studies were done using the CCB mixture cytotoxicity by brine shrimp lethality assay, antioxidant activity by DPPH assay, anti inflammatory activity by albumin denaturation assay, antimicrobial activity against Staphylococcus aureus, Streptococcus mutans, Escherichia coli and Candida albicans by agar well diffusion method, thrombolytic activity by clot lysis assay. Results: The CCB mixture showed good cytotoxic activity at varying concentration in brine shrimp lethality assay, increasing antioxidant activity with DPPH assay, increasing thrombolytic activity, increasing anti inflammatory activity with increasing concentration, the anti microbial activity was not efficient as positive controls. Conclusion: In this in-vitro study the CCB mixture shows improved cytotoxic, antioxidant, thrombolytic, anti-inflammatory and antimicrobial activity. Further studies on invivo animal and human clinical trials needs to be done.


INTRODUCTION
Plants play an important role in drug production, and the pharmaceutical industry relies heavily on natural products to produce new drugs [1,2]. According to a WHO survey, folk medicine is used by 80% of the world's population for primary health care Curcumin inhibited the development of arachidonic acid, cyclooxygenase, lipoxygenase, cytokines (interleukins and tumor necrosis factor), nuclear factor-B, and steroidal hormones. Curcumin has been shown to stabilize the lysosomal membrane and cause oxidative phosphorylation uncoupling, as well as possessing a high oxygen radical scavenging activity, which accounts for its antiinflammatory properties. Since ancient times, turmeric root has been used as a spice in India and other Asian countries. It's also commonly used as a medicine, particularly for the treatment of inflammatory conditions [8]. Over the years, curcumin has been studied for a number of medicinal purposes. It has been shown to help cure a host of illnesses, including diabetes, autoimmune disorders, and cancers [9][10][11]. Curcumin is a pleiotropic molecule that interacts with a wide range of inflammatory targets, including TNF and interleukins (ILs) [12] (Antony et al. 2021), (Sarode et al. 2021). Curcumin has also been shown to have antimicrobial action against fungi (13) and a wide range of Grampositive and Gram-negative bacteria In vitro.
Cissus quadrangularis L. is a fleshy plant found throughout the world, especially in Asia, Africa, and a few other warm tropical areas. It's one of India's most famous dishes. The whole plant is used as a digestive aid (Pachana) as well as a palliative and roborant in Ayurveda. C. quadrangularis contains a lot of ascorbic acid, carotenoids, flavonoids, and steroids. C. quadrangularis also has anti-inflammatory, antihelminthic, antifungal, antihemorrhoidal, analgesic, and antibacterial effects.
Boerhaavia diffusa L. (Nyctaginaceae) is an annual herbaceous plant found in the tropical regions of India, South America, and Africa. Jaundice, dyspepsia, nephrotic disease, convulsions, spleen enlargement, stomach pain, tension, and inflammation are all conditions that B. diffusa roots are used to treat in Ayurveda [14] (Hannah R et al. 2021), (Chandrasekar et al. 2020). Despite the fact that a methanol extract of B. diffusa (whole plant) has been shown to have antiproliferative and antiestrogenic properties [15], an ethanol extract has been shown to have hepatoprotective, immunomodulatory, and antiinflammatory properties [15][16][17].
The Nyctaginaceae family includes the herbaceous B. diffusa Linn. It can be found in the tropics and subtropics all over the world. It's been used for millennia by indigenous and aboriginal cultures, as well as in Ayurvedic or natural herbal medicine [18,19]. Pharmacological trials have shown that B. diffusa has diuretic, anti-inflammatory, antifibrinolytic [20], anticonvulsant [21], and hepatoprotective properties.
Diabetes, tension, dyspepsia, stomach pain, inflammation, jaundice, spleen enlargement, and congestive heart failure are all treated with B. diffusa in Ayurvedic medicine in India and Unani medicine in Arab countries.The aim of the study is to assess the cytotoxic, antioxidant, thrombolytic antiinflammatory and antimicrobial properties of the herbal mixture.

Collection of Plants
Curcuma longa Linn rhizomes, leaves of Cissus quadrangularis and Boerhaavia diffusa were collected in Chennai during the month of April 2021 and was washed thoroughly and shade dried. The dried plant material was finely powdered and grounded using a slender blender. The extraction was done by hydro-distillation method.

Preparation of Herbal Mixture
The materials used are plant extract, thermal electric heater, distilled water, ethanol, blood, refrigerator, petridish plates, miller hinton agar, sabouraud dextrose agar, beakers, test tube, test tube holder, ELISA plate, brine shrimps, eppendorfs. 5 grams of turmeric, 5 grams of Cissus quadrangularis, and 5 grams of Boerhaavia diffusa are combined in a beaker of 50 milliliters of ethanol and 50 milliliters of distilled water. Placed in a shaker for 24 hours. The herbal mixture was condensed and reduced to 75 ml after heating at 25-30 degrees Celsius. The herbal mixture is then purified and boiled for 10 minutes at 25 degrees Celsius. After that, the herbal solution is moved to a bottle for further examination.

Salt water preparation
Weighing 2g of iodine-free salt and dissolving it in 200 mL of purified water 10-12 ml of saline water was poured onto 6 well ELISA pots. 10 nauplii were gradually added to each well (20L, 40L, 60L, 80L, 100L). The herbal mixtures were then inserted at the appropriate concentration range. For 24 hours, the plates were incubated.
After 24 hours, the ELISA plates were examined and counted for the amount of live nauplii present, which was then measured using the formula below ( Fig. 1).
number of dead nauplii/number of dead nauplii+number of live nauplii×100 DPPH assay was used to test the antioxidant activity of the herbal mixture. Diverse concentration (2-10ug/ml) of the herbal mixture was mixed with 1ml of 0.1mM DPPH in methanol and 450ul of 50mM Tris HCL buffer (pH 7.4) and incubated for 30 minutes (Fig. 2). Later, the reduction in the quantity of DPPH free radicals was assessed dependent on the absorbance at 517 nm. BHT was employed as control. The percentage of inhibition was determined from the following equation,

Thrombolytic Activity
5 ml of venous blood drawn from a volunteer, which was placed in seven separate pre-weighed sterile microcentrifuge tubes and incubated at 37°C for 45 minutes (Fig. 3). After the clot had formed, the fluid from each microcentrifuge tube was fully released, and the clot weight was calculated by subtracting the weight of the clotcontaining tube from the weight o of distilled water was added to the microcentrifuge tubes as a negative nonthrombolytic control. The herbal mixture is added at varying concentrations. After that, all of the tubes were incubated at 37°C for 90 minutes to check for clot lysis. The released fluid was discarded during incubation, and the tubes were weighed again to see if there was a difference in weight after the clot was disrupted.

Fig. 2. Figure represents samples of herbal mixture for antioxidant study
Clot lysis value = weight of clot before mixtureweight of clot after mixture

Anti Inflammatory Activity
The herbal mixture was assessed for the antiinflammatory activity by Albumin Denaturation Assay. The mixture was tested by the following convention proposed by Muzushima and Kabayashi with specific alterations (Pratik Das et al., 2019). The herbal mixture comprising of 0.05mL of Curcuma longa Linn, Cissus quadrangularis, Boerhaavia diffusa of various fixation (10µl, 20µl, 30µl, 40µl, 50µl) was added to 0.45 mL of bovine serum albumin (1% aqueous solution) and the pH of the mixture was acclimated to 6.3 utilising a modest quantity of 1N hydrochloric acid. These samples were incubated at room temperature for 20 minutes and then heated at 55°C in a water bath for 30 minutes (Fig. 4). The samples were cooled and the absorbance was estimated spectrophotometrically at 660 nm. The standard used in this study was Diclofenac Sodium utilised as a control.
Percentage of protein denaturation was determined utilising the following equation.

Antimicrobial Activity-Agar Well
Diffusion Method

Antibacterial activity
Antibacterial activity of the herbal mixture against Staphylococcus aureus,Streptococcus mutans and Escherichia coli strains. This activity was carried out on MHA agar to establish the zone of inhibition. Muller hinton agar was prepared and sterilized at 120 pounds for 45 minutes. The media was poured onto the sterilized plates and set aside to solidify. The test species were swabbed after the wells were cut with a well cutter. The plates were filled with herbal mixture of various concentrations and incubated for 24 hours at 37°C. The zone of inhibition was assessed after the incubation period (Fig. 5)

Antifungal activity
The agar well diffusion assay uses Candida albicans as the research pathogen. The medium was prepared with Sabouraud's Dextrose Agar.
The wells were swabbed with test species and herbal mixture of various concentrations were applied to the prepared and sterilized medium. The plates were incubated for 48-72 hours at 28° C. The zone of inhibition was assessed after the incubation period (Fig. 5).

RESULTS
Brine shrimp lethality activity of the herbal mixture showed less mortality suggesting low toxicity and the control showed no mortality. The cytotoxicity assay revealed the alive nauplii at varying concentrations from 5µl to 80 µl was 10,7,9,9,9 and all the nauplii were alive in the control in Table 1. The least number of alive nauplii was found at 10 µl in Fig. 6.
A DPPH assay was done to analyse the antioxidant property which showed moderate antioxidant properties of the CCB mixture at increasing concentrations from 10µl to 50µl was 37.3, 42.6, 57.8, 77, 86.5 but the values were not more than the standard values Table 2. The significantly increasing antioxidant activity was evident as the concentration increases in Fig. 7.
Clot lysis assay was done to assess to the thrombolytic activity of the herbal mixture at varying concentrations from 200 µl to 1000 µl with the clot lysis value of 0.089g, 0.087g, 0.132g, 0.128g, 0.214g. 0.211g and 0.04 g clot lysis value for positive and negative control respectively in Table 3. The clot lysis was found to be the highest at 1000 µl which was significantly similar to the positive control in Fig. 8.
Albumin denaturation assay was done to determine the anti-inflammatory activity of the herbal mixture, the results showed the % inhibition at varying concentrations of the herbal mixture from 10µl to 50µl in 660 nm wavelength 54.6, 58.3, 68.2, 77.6, 87.3 in Table 4. The highest percentage inhibition was seen in 50µl when compared to standard in Fig. 9.
Agar well diffusion method was done to demonstrate antimicrobial activity. Antibacterial activity was done in mueller hinton agar for Staphylococcus aureus, Streptococcus mutans and Esherichia coli strains. There were no significant good results on all 3 bacterial strains. Antifungal activity was done in Sabouraud's Dextrose Agar for Candida albicans and moderate antifungal activity was found when compared to the control in Table 5. The zone of inhibition in the agar well is represented as a bar chart in Fig.  10.         Herbalists don't normally cure acute psychiatric or musculoskeletal disorders; instead, the goal of herbal medicine is to achieve long-term changes in health. Practitioners sometimes refer to treating the disease's "underlying cause," and may administer herbs to fix dysfunctional habits rather than only treating the symptoms [22].
Our study showed significantly good thrombolytic activity when compared to positive and negative control. The highest thrombolytic activity was evident at 1000 µl and the least at 200 µl. The activity was found increasing with increasing concentrations. Our study showed significantly good antioxidant properties when compared to the standard, At 50 µl concencentration the highest reading was noted as 86.5 using DPPH assay and further analysing in wavelength of 517 nm. The efficiency decreases with decreasing concentrations. The least efficiency was found in 10µl. The varying concentrations showed varying absorbance at 517 nm wavelength, at 10µl, 20µl, 30µl, 40µl, 50µl the respective absorbance were 37.3, 42.6, 57.8, 77, 86.5 respectively. The moderate cytotoxicity activity was evident in the herbal mixture.
The brine shrimp test is a rapid, economical and easy bioassay for determining the lethality of plant extracts, which correlates relatively well with cytotoxic and anti-tumor properties in most cases [23]. The majority of the time, a desired biological response is due to a combination of bioactive plant components, rather than a single component [24]. As a result, biological activity must be tested on crude extracts. The brine shrimp lethality assay has proven to be a useful tool for tracking natural product biological activities. In our study the lethality of the brine shrimp nauplii was very less. In our study good anti-inflammatory properties were evident when compared to the standard. The highest antiinflammatory effect was evident at 50µl using albumin denaturation assay. Laksmitawati et al. found that A. muricata leaf extract had anti-inflammatory activity by inhibiting the inflammatory mediators TNF-, IL-1, IL-6, and nitric oxide in an in vitro trial [28]. Maroon et al. [29] compiled a list of natural anti-inflammatory substances. Sen et al. [30] examined and tested the anti-inflammatory efficacy of the methanolic fraction of a chloroform extract of Pluchea indica roots. Goldberg et al. [31] used the mouse paw edema test to investigate the anti-inflammatory ability of aqueous extract Achillea millefolium and discovered a 35 percent reduction in oedema. Lee et al. [32] discovered that the leaves of Hedera rhombea bean have anti-inflammatory properties. Chandra et al. [33] detected antiinflammatory behaviors of ethanolic root extract of Swertia chirata (Gentianaceae) in a carrageenan-induced rat paw edema model. The anti-inflammatory effects of Sida cordifolia L. were investigated by Franzotti et al. [34] Ojewole [35] discovered that Bryophyllum pinnatum (Crassulaceae) aqueous leaf extract has antiinflammatory properties.
Bacterial infections can cause a variety of human diseases, ranging from self-limiting illnesses to potentially fatal medical conditions if left untreated. Nonetheless, antibiotic resistance is on the increase as a result of widespread use, rendering previously treatable pathogens untreatable. In the case of other infectious agents, globalization has aided the spread of (resistant) strains [36]  Inhibition zone of the ethanol extract of these plants was calculated where three were found to be sensitive against E.faecalis, C.albicans, S.aureus, S.mutans. However, the ethanol extract of Curcuma longa Linn, Cissus quadrangularis, and Boerhaavia diffusa was found to have more effective antimicrobial activity showing its maximum efficacy for both bacteria and fungi. Data from the literature, as well as our results, reveal the great potential of plant extracts as antibacterial and antifungal agents, in spite of the fact that they have not been completely investigated, more studies need to be conducted. Therefore, our results revealed the importance of plant oils when associated with antibiotics, to control resistant bacteria, which are becoming a threat to human health.

CONCLUSION
Curcuma longa Linn, Cissus quadrangularis, and Boerhaavia diffusa have also been used in the diet for centuries as flavoring agents and topical agents for a number of ailments. The improved cytotoxic, antioxidant, thrombolytic, antiinflammatory and antimicrobial activity of the CCB mixture was shown in this study. We discovered that the extract of the CCB mixture has a variety of pharmacological activities in the current research. In the current study, we discovered that the extract of the CCB herbal mixture has a range of pharmacological activities. This study has paved the way for the plant's future application in drug development and a variety of herbal mixtures with less side effects.

NOTE
The study highlights the efficacy of "HERBAL" which is an ancient tradition, used in some parts of India. This ancient concept should be carefully evaluated in the light of modern medical science and can be utilized partially if found suitable.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.