The presence of chemical constituents such as sugar, tannins, saponins, alkaloids, flavonoids, steroids, terpenoids, glycosides, phenolics, gibberellins, lipids, inositols, anthocyanidins, polysaccharides, proteins, minerals, hydrolysable tannins, and polyphenols were previously reported from R. mucronata [10]. Most of the chemical constituents were reported from the leaf and root of the plant, but the information about the metabolites isolated from the chloroform extract of the bark is rare in literature. In the present study, the following compounds were identified from the chloroform extract of the bark by the mass spectral studies.
Compound 1 (N,N′-Dicyclohexylurea): N,N′-Dicyclohexylurea was reported for the first time from the plant R. mucronata. The MS fragmentation pattern of dicylcohexyl urea is given in Fig. 5. N,N′-Dicyclohexylurea was previously reported from the plants Toddalia Asiatica [14] and Portulaca Oleraceae [15]. This compound possesses antibacterial activity against Escherichia coli [16] and acts as a ubiquinone reduction inhibitor [17] and soluble epoxide hydrolase (sEH) inhibitor [18]. sEH inhibitors may be used as a drug for the treatment of diabetes, hypertension, stroke, dyslipidaemia, pain, immunological disorders, eye diseases, and neurological diseases by increasing epoxy eicosatrienoic acid concentration and decreasing dihydroxy eicosatrienoic acids [19].
Compound 2: It is an unknown compound with the molecular formula C22H43NO, having molecular ion peak at m/z 338.3428. It shows the characteristic homologous peaks for aliphatic compound by cleaving down the chain at C-C bonds. A fragment formed at m/z 321.3164 by the cleavage of -C-N bond is due to the elimination of NH3 ([M+H]+-17.0264). Another fragment at m/z 303.3057 is due to the loss of H2O indicating the presence of hydroxyl group (-OH). The fragment at m/z 97.1017 corresponds to C7H13+ and is a characteristic feature of an aliphatic cyclic group. The proposed fragmentation pattern of the cyclic part of the compound is given in Fig. 6.
So, the compound identified is a C22 aliphatic cyclic compound with functional groups hydroxyl and primary amine.
Compound 3: [M+H]+ peak at m/z 278.1295. The molecular formula of the compound is calculated as C17H15N3O.
The fragments with m/z values 263.1060 (C16H13N3O) and 246.0789 (C16H10N2O) indicate the loss of methyl group ([M+H]+-15.0235) followed by the loss of ammonia ([M+H]+-CH6N). These fragments reveal the presence of the methyl and amine (-NH2) group in the compound. Another fragment at m/z 235.1109 (C15H13N3: [M+H]+-43.0188) resulted from the loss of -C2H3O (-CH3 and -CO) group from the molecular ion suggests the presence of -C=O moiety in the compound. The fragment at m/z 218.0844, formed by the loss of all the groups discussed above (-CH3, -NH2 and -CO) from the molecular ion, is identical to that of quindoline. Literature shows that mass fragmentation of cryptolepine leads to the formation of quindoline fragment with m/z 218.0844 by the neutral loss of -CH3 [13]. The fragment with m/z 209.0948 (C13H11N3+) formed by the loss of C4H5O (m/z 235.1109-CH≡CH) i.e. loss of C2H2-COCH3 ([M+H]+-69.0344) from the molecular ion confirms the presence of -CO group in the molecule.
The proposed structure and fragmentation of the compound is given in Fig. 7. The most remarkable result to emerge from these spectral data is the identification of a novel derivative of cryptolepine which belongs to the class of indoloquinoline alkaloid.
The indoloquinoline family of alkaloids is a very rare group of natural products. Examples are cryptolepine [20], quindoline [13], and the complex spiro-nonacyclic alkaloid cryptospirolepine [21]. Cryptolepine was reported from a shrub Cryptolepis sanguinolenta which is indigenous to West Africa and has long been used for the treatment of various fevers, including malaria [20]. So, the novel compound identified may have the potential therapeutic values which are open to further study.
Compound 4: Molecular ion [M+H]+ with m/z 258.1495. This is an unknown compound with the molecular formula C16H19NO2. Only two fragments were observed in the mass spectra, a molecular ion peak and a fragment at m/z 213.0917. The data is inadequate for the complete characterisation of the compound. The fragment formed at m/z 213.0917 (C14H13O2+) indicates the elimination of –C2H7N ([M+H]+-45.0578). From these available spectral data, the compound can be interpreted as an aromatic compound with amine functional group.
Antidiabetic activity using α-amylase inhibition assay
The chemical constituents such as glycosides, alkaloids, terpenoids, flavonoids, carotenoids, tannins, and polyphenolic derivatives are being used for diabetic treatment [22]. Of these, alkaloids are nitrogen-containing organic compounds which show antidiabetic activity either by enhancing insulin secretion from the pancreas or by reducing the blood glucose level by its transport to peripheral tissue [23]. Antidiabetic activity of alkaloids from the plants Aerva lanata, Catharanthus roseus (L.) G, and the steroidal alkaloid of Sarcococca saligna were reported in previous studies [24,25,26]. Acanthicifoline and trigonellin were the alkaloids isolated from the mangrove plant Acanthus ilicifolius [27].
R. mucronata is used as a traditional medicine for diabetes in India [2]. Antidiabetic potential studies of aqueous leaf extract of R. mucronata in the alloxan-induced diabetic rats by oral administration showed the reduction of blood glucose to normal levels. It was correlated to the presence of insulin-like protein in the extracts [28]. Antidiabetic activity of the aqueous bark extracts of R. mucronata was reported, and the proposed pathway was the glucose absorption inhibition [29].
One of the therapeutic approaches for diabetes mellitus is done by decreasing glucose absorption through the inhibition of carbohydrate hydrolysing enzymes [10]. α-Amylase is a carbohydrate hydrolysing enzymes, and its inhibition can be used as firsthand information for antidiabetic activity. The reports on α-amylase inhibition activity from chloroform fraction from the bark of R. mucronata are scarce in the literature. In the present study, α-amylase inhibition activity was assessed for chloroform fraction and the IC50 value was found to be 220.09 μg/ml. For acarbose, a standard drug that works in a similar fashion showed an IC50 of 38.40 μg/ml. The IC50 value of the extract suggests promising activity towards the α-amylase inhibition, even though its activity is less than that of acarbose. At this stage, considering only the α-amylase inhibition activity, it is difficult to arrive at any conclusion regarding the therapeutic use of the extract. Additional in vitro and in vivo animal testing and cytotoxic studies must be conducted to get more conclusions regarding the drug formulations for diabetic treatment.