In vitro antidiabetic and antimicrobial activity of Dracocephalum heterophyllum Benth. essential oil from different sites of North-western Himalayas India

Abstract Dracocephalum heterophyllum Benth (Lamiaceae) is a wild herb that possesses a number of biologically active phytomolecules. The aim of this study was to investigate comparative essential oils (EOs) composition and biological (antimicrobial and anti-diabetic) activities of D. heterophyllum from higher altitudes. Gas chromatography-mass spectrometry (GC/MS) and gas chromatography-flame ionisation detector (GC-FID) were carried out for the identification and quantification of EOs components. The hydrodistillation of fresh aerial part of D. heterophyllum gave (0.1–0.8) % w/v EOs yield. Altogether, twenty-seven constituents were identified in the among samples representing (91.0–98.2) % of the EOs composition. The β-citronellol (31.5–83.7) % contributes major constituent in all samples. The in vitro antimicrobial potential of EOs was examined against six human pathogenic bacterial and two phytopathogens fungal strains. Anti-diabetic activity exhibits excellent α-amylase and better α-glucosidase enzymes inhibitor properties. Graphical Abstract


Introduction
Dracocephalum heterophyllum Benth belongs to family Lamiaceae is an annual or perennial wild herb native from the North-western Himalaya region of higher altitude including Laddakh, Himachal Pradesh, Uttaranchal and Sikkim (Mahmood et al. 2005). It is utilised for a wide range of pharmacological properties including hepatitis, antimicrobial, antidiabetic and cytotoxic activities (Zhang et al. 2008;Arora et al. 2013;Stappen et al. 2015;Numonov et al. 2020). The aim of the present study was to isolate the essential oils (EOs) from different sites and analyse the variation in composition along with assessment of their biological activities. Furthermore, principal component analysis (PCA) was carried out on eleven major and important components of obtained EOs. Our studies explored pharmacological properties of D. heterophyllum EOs against three gram-positive and three gram-negative bacteria as well as two fungal phytopathogens Fusarium graminearum and Fusarium oxysporum. This work also showed inhibition activities against a-amylase and a-glucosidase which are key enzymes linked to the pathogenesis of diabetes.

Essential oil composition
Every individual year study in 2018 and 2019 that was based on the different sites (Table S1) was carried out within about 182-and 32-kilometers road side range of the Ladakh and Himachal Pradesh ( Figure S1, Supplementary material) respectively. The hydrodistilation of the fresh leaves of D. heterophyllum gave (0.1-0.8) % yield of EOs ( Figure S1, Supplementary material). The obtained EOs were analysed by gas chromatography-flame ionisation detector (GC-FID) and GC-MS (gas chromatography-mass spectrometry). The GC-MS data accounted for identification of twenty-seven constituents in the all EOs samples constituting (91.0-98.2) % of the total oil composition (Table S2, Supplementary material). The EOs were primarily characterised by oxygenated monoterpenes (80.4-96.5) % followed by monoterpene hydrocarbons (0.4-6.8) %, sesquiterpene hydrocarbons (0.3-2.4) %, oxygenated sesquiterpene (0.1-0.5) % and other (0.1-3.0) %. The major components of the EOs were identified as b-citronellol (31.5-83.7) %, citronellyl acetate (1.8-10.3) % and citronellal (0.9-4.3) %. The previous literature also agreed that the b-citronellol was the major component in the D. heterophyllum used in perfumes (Mahmood et al. 2005;Singh et al. 2008;Kaul et al. 2010;Stappen et al. 2015). However, 1,8-cineol reported as the major constituent in the D. heterophyllum EOs collected from Gan-Nan region of China (Zhang et al. 2008). Two years comparative studies of the same sites (D-1A, D-1B, D-4A, D-4B and D-5A, D-5B) showed the reproducibility with a minor difference (0.1) % in EOs yield and chemical composition. The classification of EOs based on principal component analysis (PCA) of eleven most common and important components score plot showed three clusters of all samples with their loading plots PC-1 & PC-2 summarised in Figure S2 (Supplementary material).

Antimicrobial activity
The antibacterial plate assay confirmed a strong activity of D. heterophyllum EOs against both human pathogenic gram-positive (Bacillus subtilis MTCC 121, Staphylococcus aureus MTCC96, Listeria monocytogenes MTCC 1144) and gram-negative (Escherichia coli MTCC 43, and Klebsiella pneumoniae MTCC 109) bacterial groups. Among the six bacteria, L. monocytogenes, S. aureus, E. coli and K. pneumoniae are the four most susceptible to all samples, with the exception of sample D-4A in L. monocytogenes. In case of B. Subtilis sample D-6A and D-3B to D-6B showed significant susceptibility towards EOs. Whereas Pseudomonas aeruginosa MTCC 2453 couldn't show susceptibility towards any of the samples. The results of zone of inhibition (ZOI), minimum inhibitory concentration (MIC), and percentage mycelial growth inhibition are summarised in Table S3 and S4 (Supplementary material). In antifungal activity complete mycelial growth inhibition was observed against both plant pathogenic fungal strains, i.e., Fusarium graminearum MTCC 1893 and Fusarium oxysporum MTCC 284 at concentration 5 mL/mL as shown in Figure S3 (Supplementary material). The activity of D. heterophyllum EOs against B. subtilis, S. aureus, E. coli and P. aeruginosa agreed with literature reported studies (Zhang et al. 2008;Stappen et al. 2015). Gram-positive bacteria have extremely impermeable cell wall construction, so they demonstrated higher vulnerability to EOs than gram-negative bacteria (Nazzaro et al. 2013). Potential synergistic as well as antagonistic effects were considered during the evaluation of EOs biological activities (Toroglu 2011). The observed antimicrobial activity of the EOs was mainly due to the presence of oxygenated monoterpene, especially b-citronellol in a higher amount. In previous studies, b-citronellol identified as an active antimicrobial molecule Kotan et al. 2007;Guimaraes et al. 2019).

Enzyme inhibitory activities
In relation with diabetes (Lebovitz 1984) both a-amylase and a-glucosidase enzymes were shown to be inhibited by D. heterophyllum EOs in a concentration-dependent manner and their half-maximal inhibitory concentration (IC 50 ) values were reported (Table S5, Supplementary material). In comparison to positive control a-amylase inhibitor with an IC 50 value of (2.4 ± 0.2) lg/mL, the most promising results were obtained against a-amylase with an IC 50 value of (0.7 ± 0.0 to 3.9 ± 0.3) lg/mL. In contrast to the positive control acarbose, IC 50 value (0.8 ± 0.3) lg/mL, the a-glucosidase inhibited with an IC 50 value of (2.8 ± 0.1 to 4.5 ± 0.3) lg/mL ( Figure S4, Supplementary material). According to prior research investigations components of EOs have both a synergetic and antagonistic action, resulting in a wide range of results (Dang et al. 2016).

Conclusion
This study discloses that the EOs extracted from different sites had variation in the yield and composition whereas same sites consecutive studies showed the reproducibility in the result with b-citronellol as a principal component. The acquired results of PCA support the concept that the EOs extracted from collected samples of different sites may be affected by the local environmental conditions. Furthermore, because of its promising display of antimicrobial, a-amylase and a-glucosidase activities shown here, these EOs extracted from different sites can also be considered for further pharmacological studies related to develop as new natural products.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
Authors are grateful to Director, CSIR-IHBT Palampur (H.P.), India, for continuous encouragement and support. We are also grateful to NMHS project of MoEF&CC sanction no. GBPNI/NMHS-2018-19/SG/178 and CSIR, New Delhi for financial support (project nos. HCP-0007 and MLP-0142) and for fellowship to Mr. Ram Chander.