Exploring The In Vitro Anti-Inflammatory and Hypoglycemic Effects of Bunchosia Argentia Leaf Extracts

Medicinal plants have been used for centuries in traditional healing practices and ethnomedicinal systems across the globe¹. According to WHO estimates, approximately 75% of the global population relies on herbs and other forms of traditional medicine for treating various ailments. Indian traditional medical systems such as Ayurveda, Siddha, and Unani largely depend on the use of plant-based materials². Medicinal products derived from plants have long drawn global scientific interest because of their minimal side effects and beneficial impact on human health³. Plant-based medicines have played a crucial role both as therapeutic agents and as essential raw materials in the production of various traditional and modern drugs. The use of medicinal plants for treating illnesses and ailments likely dates back to the earliest stages of human civilization. Medicinal plants naturally produce and store various secondary metabolites such as alkaloids, sterols, terpenes, flavonoids, saponins, glycosides, cyanogenic compounds, tannins, resins, lactones, quinones, and essential oils⁴.

Bunchosia argentea known as silver peanut butter fruit is a species of flowering plant in the acerola family, Malpighiaceae. The plant is a native to the north western South America (Colombia, Ecuador, Bolivia, Venezuela, Brazil and Peru). Also known as Bunchosia fruit tree, it is the plant that is grown as an ornamental for its vibrant yellow blooms and red fruit which produces simultaneously. Some of the popular common names of the plant are Bunchosia, Green Plum, Monk’s Plum, Peanut Butter Fruit and Peanut Butter Tree. The name Bunchosia is said to come from an Arabic word, bunchos meaning coffee, for the resemblance of the seed-containing pyrenes of the fruit to the mericarps of coffee Arabica. It is also called peanut butter fruit because of its texture, but not its taste, that is similar to peanut butter.

PLANT PROFILE

Table no.1 Taxonomical classification of Bunchosia argentea

PLANT DESCRIPTION

Peanut butter fruit is a small, highly ornamental and hardy tropical, evergreen perennial tree or shrub that grows about 2-4m tall. The plant is found growing in dry and moist limestone forests near the coast, normally prefers moist, fertile loamy soil rich in organic matter.

Stem : They are persistently sericeous, the older woody stems are glabrate. The tree is stiff, spreading branches.

Leaves : Short petiolate leaves are simple, opposite, lanceolate to ovate, chartaceous, 10-27cm long and 5-10 cm wide, rounded to wedge-shaped, marginally striped, spiky, sparsely scaly on both sides, has 6-7 lateral veins.

Fig 1: Morphology of Bunchosia argentea leaves

Flowers: Carrying numerous yellow hermaphrodite flowers, on a 0.5-1 cm long pedicel provided with glandular tubercles, with penta partid calyx, compact axillary racemose inflorescences, 5 ungiillated petals with 5-6mm long, with an oval laminate and fringed serrated margin. The peanut butter plant will go through several blooming cycles from March until October.

Fig 2: Morphology of Bunchosia argentea flower

Fruits : Fruits are borne in clusters. They are ovoid, 2.5cm long, ellipsoid to obovate berry with thin pale green skin turning to orange then red at maturity. The internal pulp of the peanut butter fruit is very thick and slightly sticky similar to that of soft persimmon and surrounds a large central seed.

Fig 3: Morphology of Bunchosia argentea ripen fruit

TRADITIONAL USES

Eating peanut butter help to reduce the problems of heart as it reduces the cholesterol levels which is the main reason behind heart ailments. Not only it solves heart problems but also makes our nerves function effectively⁵.

ANTI-INFLAMMATORY ACTIVITY

Inflammation is a complex biological response that serves to protect and repair tissues following injury caused by mechanical damage, infections, or autoimmune triggers. It can manifest as either an acute, short-term reaction or a chronic, long-lasting condition⁶. Inflammation is triggered by various factors such as mechanical injury, autoimmune reactions, or infections. It can be categorized as either acute or chronic. Chronic inflammatory conditions, such as rheumatoid arthritis, continue to pose significant health challenges worldwide. Although synthetic drugs currently dominate the market for inflammation treatment, their long-term use is often associated with serious side effects, including gastrointestinal bleeding and peptic ulcers. Therefore, there is a pressing need to develop new anti-inflammatory agents that are both safe and effective. In this context, the search for plant-based anti-inflammatory compounds has become a major focus in scientific research within the field of herbal medicine⁷. NSAIDs (Non-Steroidal Anti-Inflammatory Drugs) are among the most widely used medications globally for the treatment of inflammation and related conditions. Most NSAIDs are carboxylic acid-containing compounds, including salicylate derivatives (such as aspirin), heterocyclic and carboxylic acid derivatives (like indomethacin), propionic acid derivatives (such as ibuprofen, ketoprofen, and flurbiprofen), and phenylacetic acid derivatives (like diclofenac). These drugs exert their anti-inflammatory effect by binding to the active site of the cyclooxygenase (COX) enzyme, thereby blocking the access of arachidonic acid and inhibiting the COX pathway⁸. The serious side effects associated with both steroidal and non-steroidal anti-inflammatory drugs have prompted the search for new anti-inflammatory agents derived from natural botanical sources, which are expected to offer therapeutic benefits with fewer adverse effects⁹.

Studies have shown that bioactive compounds found in medicinal plants—such as alkaloids, saponins, phytosterols, tannins, and flavonoids—exhibit significant anti-inflammatory properties. Several mechanisms have been proposed to explain the anti-inflammatory effects of these plant-derived compounds, including:

1. Inhibition of 15-Lipoxygenases (15-LOX):
Lipoxygenase enzymes (including 5, 8, 12, and 15-LOX) play crucial roles in various inflammatory processes. Among them, 15-LOX is involved in the synthesis of leukotrienes from arachidonic acid. Since leukotrienes are potent mediators of inflammation and allergic responses, inhibiting 15-LOX activity is considered a promising therapeutic approach in managing inflammatory conditions.

2. Inhibition of Nitric Oxide Synthase (NOS):
Although not a universal feature of all plant flavonoids, some have been found to suppress the production of nitric oxide (NO) by downregulating the expression of inducible nitric oxide synthase (iNOS). Flavones and their amino-substituted derivatives have particularly shown potential in inhibiting NO production.

3. Inhibition of Cyclooxygenases (COX):
Flavonoids, a class of polyphenolic compounds, can inhibit prostaglandin biosynthesis by targeting cyclooxygenase enzymes. Both COX-1 and COX-2 isoforms are implicated in inflammation, and many plant extracts and phytochemicals have demonstrated inhibitory activity against these enzymes, contributing to their anti-inflammatory effects.

4. Inhibition of Phospholipase A2 (PLA2):
Phospholipase A2 releases arachidonic acid from membrane phospholipids, initiating the production of eicosanoids such as prostaglandins, thromboxanes, and leukotrienes. By inhibiting PLA2, plant-based agents can effectively block both the COX and LOX pathways, disrupting the inflammatory cascade and providing relief from inflammation.

5. Suppression of Pro-inflammatory Cytokines:
Certain plant-derived compounds can inhibit the production or activity of pro-inflammatory cytokines, which play key roles in amplifying the inflammatory response either directly or by inducing the expression of adhesion molecules and other cytokines in target cells.

6. Modulation of Pro-inflammatory Gene Expression:
Herbal compounds often affect cellular regulatory pathways by targeting protein kinases involved in signal transduction, such as protein kinase C and mitogen-activated protein kinases (MAPKs). Inhibiting these enzymes alters the DNA-binding activity of key transcription factors like nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1), ultimately reducing the expression of genes involved in inflammation.

These mechanisms highlight the potential of medicinal plants as sources of novel anti-inflammatory agents with multiple molecular targets and fewer side effects compared to conventional drugs¹⁰.

Fig 4: Mechanism of anti-inflammatory activity on medicinal plants

HYPOGLYCEMIC ACTIVITY

Diabetes mellitus is an increasingly prevalent global health concern, placing a significant financial burden on healthcare systems and posing serious challenges for medical policy development. It is one of the most common endocrine metabolic disorders, characterized by impaired glucose homeostasis and disturbances in carbohydrate, fat, and protein metabolism due to defects in insulin secretion, insulin action, or both.

The human body is equipped with both enzymatic and non-enzymatic antioxidant defense mechanisms that help reduce the generation of reactive oxygen species (ROS), which are implicated in the development of numerous degenerative diseases, including diabetes. The incidence of diabetes is rising rapidly across all regions of the world¹¹.

Among the various types of diabetes, type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) are the most widely studied. T1DM, also known as insulin-dependent diabetes, is typically caused by autoimmune destruction of pancreatic islet beta cells, leading to insufficient insulin production. It commonly manifests in children and adolescents. In contrast, T2DM—also known as non-insulin dependent diabetes—is primarily associated with insulin resistance and impaired insulin utilization, resulting in persistent hyperglycemia. T2DM accounts for approximately 90–95% of all diabetes cases worldwide¹².

According to the World Health Organization (WHO), the global diabetic population is expected to exceed 300 million by the year 2025. Furthermore, the WHO projects that diabetes will become the seventh leading cause of death by 2030.

Current therapeutic options for diabetes include insulin and various classes of oral hypoglycemic agents, such as sulfonylureas, biguanides, and glinides. However, many of these treatments are associated with significant adverse effects, highlighting the need for safer and more effective alternatives.

Traditional medicine, particularly plant-based remedies, holds significant promise for the development of new antidiabetic drugs. Many medicinal plants contain bioactive compounds such as glycosides, alkaloids, terpenoids, flavonoids, and carotenoids, which have demonstrated antidiabetic potential. The hypoglycemic effects of several such plants have been validated, and ongoing research is focused on elucidating the mechanisms underlying their antidiabetic activity¹³.

Fig 5: Medicinal plants with multiple effect on DM.

MATERIALS AND METHODS

PLANT COLLECTION AND AUTHENTICATION

Bunchosia argentea, a flowering plant grows as a perennial tree or shrub. Fresh leaves of Bunchosia argentea were collected in the month of November from North Paravoor, Ernakulam district of Kerala. Parts including bud, flower and leaves was taken to SD College, Alappuzha for authentication. The plant was authenticated as Bunchosia argentea (Jacq.) DC. of Malpighiaceae family by Dr. Jose Mathew, Assistant professor, Department of Post Graduate Studies & Research in Botany, SD College.

EXTRACTION

Maceration was a popular and inexpensive homemade technique for the preparation of tonic since a long time. Moreover, this technique was used for the extraction of essential oils and active compounds from plant materials. Generally, the maceration procedure consists of multiple steps in extraction. The whole or coarsely powdered crude drug undergoes grinding to increase the surface area for proper mixing of powdered materials with the solvent. This process is done in a closed vessel where an appropriate solvent (menstruum) is added. Next, the solvent is strained off followed by pressing the solid residue of the extraction process known as marc to recover an optimum amount of occluded solution. Both the obtained pressed out liquid and the strained solvent are mixed together and separated from unwanted materials by filtration. Frequent agitation during maceration facilitates extraction by two processes: (1) promotes diffusion, (2) separates concentrated solution from the sample surface by adding new solvent to the menstruum for increasing the extraction yield¹⁴.

PROCEDURE

The collected leaves material was shade dried and coarsely powered using mechanical grinder. The aqueous and alcoholic extract was prepared from the leaves of Bunchosia argentea separately. The coarsely powdered leaves were subjected to the process of maceration by 7 days with water and ethanol respectively at room temperature. After, was filtered, concentrated. Both the crude extract was stored at 4?C separately¹⁵.

PHYTOCHEMICAL SCREENING

The crude samples were subjected to qualitative chemical test for the detection various   plant   constituents   like   amino   acids, proteins, glycosides, saponins, triterpenoids, flavonoids, carbohydrates, alkaloids, tannins.

Carbohydrates

Tannins

General test for the presence of Glycosides

a. Test A: Extract 200mg of the powdered drug material with 5ml of dilute (10%)  sulphuric acid by heating in a test tube on a water bath for 2min. Centrifuge or  filter, pipette off supernatant or collect the filtrate. Neutralize the acidic extract  using alkaline such as Sodium hydroxide (5%). Add 0.1ml of Fehling’s reagent A  and B and heat on water bath for 2min. Observe the quantity of red precipitate formed and compare it with that formed in Test B.

b. Test B: Extract 200mg of drug material using 5ml of distilled water instead of sulphuric acid, after boiling add equal volume of water to that of the sodium hydroxide used in the above test. Add 0.1ml of Fehling’s reagent A and B until alkaline and heat on a water bath for 2min. Note the quantity of red precipitate  formed.

Saponin glycosides

                    Table no 1: Phytochemical screening of Bunchosia argentea

PROCEDURE

ANTI-INFLAMMATORY ACTIVITY

HRBC membrane stabilization method

The collected blood was mixed with equal volume of sterilized Alsever medium (2%w/v dextrose, 0.8% sodium citrate, 0.5.5 citric acid, and 0.42% sodium chloride in water). The blood was centrifuged at 3000rpm for 10min, and packed cell were washed with isosaline (0.85%, pH7.2) and finally 10%v/v suspension was made with isosaline. The assay mixture contained secondary metabolites from the plant extract, 1ml phosphate buffer (0.15M pH 7.4), 2ml hyposaline (0.36%) and 0.5ml HRBC suspension. Diclofenac was used as reference drug. Instead of hyposaline, 2ml of distilled water was used as control. The assay mixtures were incubated at 37?c for 30 minutes and centrifuged at 3000rpm for 10 minutes. The HB content in the supernatant was estimated using UV-vis spectrophotometer at 560nm¹⁷.

HYPOGLYCEMIC ACTIVITY

Glucose uptake by yeast cell method

PROCEDURE

Commercial baker’s yeast was dissolved in distilled water to prepare 1% suspension. It is kept overnight at room temp.(25?c). On next day, yeast cell suspension was centrifuged at 3000 rpm for 5 mins. The process was repeated by the addition of distilled water until a clear supernatant was obtained. Exactly 10 parts of the clear supernatant fluid were mixed with 90 parts of distilled water to get a 10% v/v yeast cell suspension. About 1-5 mg w/v of plant extract was mixed with DMSO till dissolution. The mixture was then supplemented with various concentration (5,10,25Mm) of 1 ml of glucose solution and incubated for 10 minutes. To initiate the reaction, 100µl of yeast suspension poured in the mixture of glucose and extract vortexed and incubated for 60 mins at 37?c. After incubation, the tubes are centrifuged for 5 mins at 3800rpm and glucose was estimated by using a spectrophotometer(uv5100B) at 520nm. Absorbance for respective control was also recorded on same wavelength. Where control is the solution having all reagents excepts the test sample. Metformin was standard drug¹⁸.

RESULT AND DISCUSSION

• 90g of dried leaf powder of Bunchosia argentea yielded 19.87g of crude aqueous 
  extract which was about 22.07% w/w.
• 90g g of dried leaf powder of Bunchosia argentea yielded 7.84 g of crude ethanol
  extract which was about 8.71 % w/w.

Fig 6: Percentage yield of different extract of Bunchosia argentea leaves.

Preliminary phytochemical screening of crude aqueous and ethanol extract of Bunchosia argentea leaves shows the presence of following constituents.

        (+) indicate active constituents in high amount

ANTI-INFLAMMATORY ACTIVITY 

                    Table no 2:  Percentage protection for anti-inflammatory activity

Fig 7: The effect of aqueous and ethanolic extract of Bunchosia argentea by HRBC method. The percentage protection was measured in the presence of various concentrations of Bunchosia argentea and Diclofenac sodium.

HYPOGLYCEMIC ACTIVITY

Table no 3: Percentage glucose uptake for hypoglycemic activity

Fig 8: The effect of aqueous and ethanolic extract of Bunchosia argentea by HRBC method. The percentage protection was measured in the presence of various concentrations of Bunchosia argentea.