Anti-inflammatory and anti-diabetic properties of plant extracts and compounds from Scabiosa columbaria and Sclerocarya birrea

Abstract

Given that South Africa is home to around 10% of all flowering plant species known to humans, the country is blessed with an abundance of natural resources. About 24,000 plant species have yet to be fully uncovered for the benefit of humanity, making this a significant resource. The use of traditional medicine to cure illnesses is still widespread in South Africa despite the country's rapid urban and infrastructure development, more westernization, and accessibility to typical western medical institutions. Intricately woven within South African culture, the use of medicinal plants to heal illnesses is still prevalent. Additionally, due to Africa's failing healthcare system, using medicinal plants for health reasons is a well-accepted alternative that is practiced by all races, classes, and socio-economic classes. Bio-active extracts, fractions, and compounds have been identified as effective treatments for inflammatory and diabetic disorders based mostly on ethnomedicinal and empirical knowledge on traditional applications of plants. South Africa is a country with a high plant diversity of over 30,000 species of higher plants and 3,000 of these plant species have been found to be used in the traditional medicine for anti-inflammatory purposes. Inspired by the traditional uses of plant species, various scientists have studied the anti-inflammatory activities of South African plant species.

Current cosmeceutical and drug discoveries rely on the massive screening of natural product libraries against various extracellular and intracellular molecular targets to find novel chemotypes with the desired mode of action. In the pharmaceutical and cosmeceutical industries, there is an increasing interest in the demand for natural ingredients with potential health benefits, such as anti-inflammatory, anti-diabetic, and anti-cancer properties, in an effort to replace or lessen the usage of synthetic products. With 60% of marketed pharmaceuticals and cosmeceuticals, natural ingredients have long been a key source of medicinal and cosmeceutical scaffolds. Nature continues to prove to be a source of new bioactive molecules with high safety profiles, despite the fact that many synthetic chemists are focused on synthesizing potent compounds with high toxicity profiles for pharmaceutical and cosmeceutical goals. Therefore, natural product chemists keep looking for novel leads. As a continuation of these efforts, this study aimed to identify and develop new natural anti-inflammatory ingredients from selected South African plant species for commercial application in different market sectors based on their traditional uses and literature data and to isolate and characterize biologically active compounds using modern hyphenated analytical techniques from biologically active plant species.

A literature survey was carried out to identify South African plants based on their traditional uses to include in this study. A scoring system was applied to rank them and 3 plant species (Scabiosa columbaria, Commiphora pyracanthoides and Pelargonium capitatum) belonging to three families were selected. The 3 plant species were collected from the University of Pretoria Experimental farm, KwaZulu Natal and Limpopo and extracted singly using cosmetic acceptable solvents (acetone, ethanol water/ethanol (1:1) and water). The extracts were tested in different assays (anti-inflammatory and skin even tone). After testing different extracts of the plant species, the ethanol extract of S. columbaria roots was selected for further evaluation to identify the compound/s responsible for the anti-inflammatory activity and development as a potential active herbal ingredient based on good anti-inflammatory efficacy data and no anti-inflammatory reports in the literature.

UPLC-QTOF-MS analysis of the ethanol extract of S. columbaria roots led to the tentative identification of fifteen compounds which are loganic acid (53) scrophuloside A1 (peak 2), 3,4-dicaffeoylquinic acid (peak 3), cantleyoside (peak 4), sylvestroside III (peak 5), triplostoside A (54), hederagenin (55), maslinic acid (peak 8), 2-isoursolic acid (56), glycyrrhetaldehyde (peak 10), pomaceic acid (57), euscaphic acid (58) and 3-oxoglycyrrhetinic acid (61).

The presence of loganic acid (62), cantleyoside -dimethyl-acetal (63), ursolic acid (64), 2-isoursolic acid (65), 24-nor-2α,3β-dihydroxyolean-4(23),12-ene (66) and hederagenin (67) in the ethanol extract of S. columbaria roots was confirmed by isolation and structure elucidation of the compounds using MS and NMR data. Significant reduction of nitric oxide levels in RAW 264.7 macrophages was observed for ursolic acid (64) (12.5, 25 and 50 µg mL-1; 0.0702, 0.0558 and 0.0357 µg/mLrespectively), 24-nor-2α,3β-dihydroxyolean-4(23),12-ene (66) (12.5, 25 and 50 µg mL-1; 0.0543, 0.0327 and 0.0231 µg/mLrespectively) and hederagenin (67) (12.5 and 25 µg mL-1; 0.0735 and 0.0513 µg/mLrespectively) compared to the positive control aminoguanidine (12.5 µg mL-1; 0.0336 µg mL-1). At a concentration of 25 and 50 µg mL-1, 24-nor-2α,3β-dihydroxyolean-4(23),12-ene (66) demonstrated a potent reduction in nitric oxide level in RAW 264.7 macrophages. The compounds identified in the ethanol extract of S. columbaria roots will be used as chemical markers for quality control purposes, for batch-to-batch reproducibility that is required for commercializing the herbal ingredient. The anti-inflammatory activity of 24-nor-2α,3β-dihydroxyolean-4(23),12-ene (66) has been reported for the first time in this work. The active compounds (ursolic acid (64), 24-nor-2α,3β-dihydroxyolean-4(23),12-ene (66) and hederagenin (67)) were structurally similar and contained a β-hydroxy group at C-3. The compound 2- isoursolic acid (65) was inactive and had a hydroxy group at C-2 instead of C-3, which suggests that the position of the β-hydroxy group may play a role in the nitric oxide inhibition activity. A concentrated form of the ethanol extract of S. columbaria roots can be developed to have a higher concentration of the active compounds for commercial application as an anti-inflammatory ingredient.

Diabetes is a global health problem and a national economic burden. Although there are many anti-diabetic medications on the market, there is still a need for innovative treatment agents with increased efficacy and less side effects. Because they are more diverse and have minimal side effects than synthetic medications, pharmaceuticals made from natural products are more appealing. In line with this quest, the Department of Science and Innovation (DSI) established the African Traditional Medicines collaboration, where research was carried out to find and create a new natural anti-diabetic ingredient. S. birrea was chosen because its leaves and stem bark have historically been used to treat conditions including diabetic mellitus. Although type-2 diabetes mellitus was reportedly inhibited by S. birrea leaf extract, the compounds responsible for the anti-diabetic activity have not yet been identified, and their discovery will be helpful for commercial use. In order to discover the chemical compounds in S. birrea that are responsible for the anti-diabetic activity and to employ them as chemical markers for quality control purposes, this Ph.D. research was carried out.

Of all the extracts of S. birrea leaves tested, aqueous extract 4 showed statistically significant activity including at the lowest test concentration (0.01 µg mL-1) and was selected to isolate and identify the compounds responsible for the anti-diabetic activity (glucose uptake activity).

UPLC-QTOF-MS analysis of the spray-dried aqueous leaf extracts of S. birrea (aqueous extracts 1 and 4) led to the tentative identification of sixteen compounds which are quinic acid (peak 1), gallic acid (peak 2), procyanidin B2 (peak 3), gallocatechin (peak 4), Pistafolin A (peak 5), epicatechin (peak 6), myricetin-3-O-β-D-glucuronide (88), gossypin (peak 8), quercetin-3-O-(6''-galloyl)-β-D-glucopyranoside (peak 9), myricetin-3-O-α-L-rhamnopyranoside (peak 10), quercetin-3-O-β-D-glucuronide (89), quercetin-3-O-arabinoside (peak 12), quercetin-3-O-α-L-rhamnopyranoside (peak 13), kaempferol-3-O-α-L-rhamnopyranoside (peak 14), myricetin (90) and quercetin (peak 16). The presence of myricetin (91), myricetin-3-O-β-D-glucuronide (92) and quercetin-3-O-β-D-glucuronide (93) in the aqueous leaf extract of S. birrea was confirmed by isolation and structure elucidation of the compounds using MS and NMR data. Myricetin-3-O-α-L-rhamnopyranoside (peak 10), gallic acid (peak 2), quercetin-3-O-arabinoside (peak 12) and quercetin-3-O-α-L-rhamnopyranoside (peak 13) were previously reported to occur in S. birrea. Quinic acid (peak 1), myricetin (90) and quercetin (peak 16) were previously reported to have anti-diabetic activity. Myricetin-3-O-β-D-glucuronide (92) and quercetin-3-O-β-D-glucuronide (93) have not been previously reported to occur in S. birrea. Myricetin (91), myricetin-3-O-β-D-glucuronide (92) and quercetin-3-O-β-D-glucuronide (93) significantly increased the glucose uptake in differentiated C2C12 myocyte cells at different test concentrations; myricetin (91) (0.1 and 10 µg mL-1; 85.7 and 109.1%, respectively), myricetin-3-O-β-D-glucuronide (92) (0.1 and 10 µg mL-1; 61.6 and 88.8%, respectively) and quercetin-3-O-β-D-glucuronide (93) (0.1 and 10 µg mL-1; 40.9 and 43.9%, respectively) compared to the treatment of insulin (0.1 µM; 100%). At a concentration of 10 µg mL-1, myricetin (91) demonstrated both a potent and concentration-dependent stimulatory action on glucose uptake in the C2C12 myocytes, matching that of insulin, the positive control. Myricetin-3-O-β-D-glucuronide (92) has not been previously reported to have anti-diabetic activity, and the combination of this compound with other known anti-diabetic compounds in S. birrea contributes to the plant’s anti-diabetic efficacy. These anti-diabetic compounds will be used as chemical markers for quality control purposes required for commercializing the herbal ingredient. This study provides scientific data to support the commercial application of the aqueous extract of S. birrea leaves as an anti-diabetic ingredient.

By finding novel active components and molecules responsible for the biological efficacy to be exploited as chemical markers for commercial application, the project's ultimate purpose was accomplished. The indigenous knowledge on the use of medicinal plants proves to be useful for identifying ingredients and developing products for the various market sectors, and this research provides scientific evidence on the value of South Africa's plant biodiversity as a continuing source of biologically active ingredients. Secondly the bioassay guided isolation method used in the isolation of active compounds proved to be a useful technique with the bioassay acting as a specific detector at every purification step. Though it can be criticized for being time consuming and resource intensive compared to the conventional method of isolation, the approach compensates for these shortcomings by its effectiveness.