Assessment of chemically characterized Salvia sclarea L. essential oil and its combination with linalyl acetate as novel plant based antifungal, antiaflatoxigenic and antioxidant agent against herbal drugs contamination and probable mode of action

Abstract The present investigation reports antifungal and antiaflatoxigenic efficacy of Salvia sclarea essential oil (SSEO) and its combination with Linalyl acetate (LA) (1:1) against herbal drug deteriorating molds and aflatoxin B1 contamination. GC-MS analysis of SSEO showed Linalyl Acetate (LA) (61.33%) and Linalool (LL) (17.59%) as major components. The SSEO and LA combination displayed better antifungal and antiaflatoxigenic activity as compared to SSEO and LA used individually. SSEO and LA combination was effective in reduction of ergosterol and enhanced leakage of vital ions and UV-absorbing materials in a dose dependent manner. The combination caused significant reduction in cellular methylglyoxal content, an aflatoxin inducer suggesting its future application for development of aflatoxin resistant herbal drug varieties through green transgenics. The combination also showed pronounced antioxidant activity as compared to SSEO and LA used separately. Interestingly, the combination showed significant in situ protection of Picrorhiza kurroa rhizomes against mould infestation. Graphical Abstract


Introduction
Herbal drugs have a long history in human disease treatment and are used globally even in the era of modern medicine. The contamination of harvested herbal raw materials by bacteria, and toxigenic molds, diminishes their medicinal efficacy (Dubey et al. 2008). Inappropriate collection and harvesting practices and changes in moisture and pH favor sporulation of Aspergillus flavus as well as toxic aflatoxin B 1 (AFB 1 ) secretion in drug samples which may cause serious human health disorders. Furthermore, mold infestation of herbal drugs also alters the bioactive components, thereby affecting the medicinal potency.
In view of harmful effects of synthetic chemicals, botanical preservatives are currently being preferred as safer alternatives to inhibit fungal growth and aflatoxin secretion. Among different botanical preservatives, essential oils and their formulations are of immense importance due to their volatile nature, less or no residual toxicity and high safety profile to human and other mammals (Galvez et al. 2018).
Salvia sclarea L. is commonly growing herb (family Lamiaceae). The essential oil (EO) is widely used as analgesics, anti-inflammatory, anti-viral and anti-mutagenic agents along with utilisation in food and cosmetics.
The present work was focused to determine the principle components of EO and evaluation of efficacy of intact oil and its combination with Linalyl Acetate (LA) against AFB 1 secretion and probable antifungal and antiaflatoxigenic mode of action. Moreover, activity of Salvia sclarea essential oil and LA combination on cellular methylglyoxal (AFB 1 inducer) level was also investigated for recommending its use as natural herbal drug preservative 2. Results and discussion 2.1. Mycoflora analysis of herbal drug samples and selection of toxigenic strain pH of herbal drug samples ranged between 4.97-5.51 (Supplementary material Table S1). In this pH range, occurrence of Aspergillus, Penicillium and other biodeteriorating species are predominant which produce different hydrolytic enzymes responsible for degradation of active chemical constituents of herbal drugs (Gautam et al. 2009). Moisture content of samples ranged between 14.73% and 17.81% (Supplementary material Table S1). High moisture content is known to support the prolific growth of molds and their associated mycotoxins. Eighteen species of storage fungi were recorded during mycoflora analysis of herbal raw materials. Among different fungal isolates, per cent occurrence frequency of A. flavus was the most prominent in all samples except Withania somnifera (Supplementary material Figure S1). During toxigenicity assay of different A. flavus isolates, the toxin content ranged from 80.15 to 2028.90 mg/L and Af LHP PK 6 was found to produce maximum AFB 1 (2028.90 mg/L) (Supplementary material Table S2).

Chemical characterization of SSEO
GC-MS analysis of SSEO identified 33 components representing 94.31% of total EO. The relative proportion of identified components and their retention times are presented in Supplementary material Table S3. LA (61.33%) was found as dominant component followed by linalool (17.59%) and c-hotrienol (2.48%). The presence of LA and linalool as major components of SSEO has also been reported earlier by Raafat and Habib (2018) and Azizkhani et al. (2016). However, caryophyllene oxide (24.1%), sclareol (11.5%), spathulenol (11.4%), 1H-naphtho (2,1,6) pyran (8.6%) and b caryophyllene (5.1%) as the most abundant component has been described by Yuce et al. (2014). Such variations in major compounds and chemical compositions may results from differences in climatic conditions, soil characteristics, geographical conditions and method of essential oil extraction (Burt 2004).
2.3. Effect of SSEO and its LA (1:1) combination on mold species growth and AFB 1 secretion SSEO showed complete inhibition of Af LHP PK 6 strain at 5.0 mL/mL concentration while LA at 1.5 mL/mL concentration (Supplementary material Table S4). However, the combination of SSEO and LA (1:1) demonstrated better efficacy (1.0 mL/mL) than SSEO and LA used separately. This may be because of presence of more amount of LA in the combination. Minimum aflatoxin inhibitory concentrations (MAIC) of SSEO, LA and its combination were recorded as 3.0, 1.0 and 0.75 mL/mL, respectively (Supplementary  material Table S4). It was observed that, MAIC was far lower than the MIC (minimum inhibitory concentration). Improved antiaflatoxigenic efficacy of LA and its combination with SSEO may be due to the inhibition of sporulation, reduced conidia germination and altered carbohydrate catabolism in Af LHP PK 6 cells (Bluma et al. 2008). The combination showed broad range fungitoxicity against different common drug deteriorating moulds except Aspergillus nidulans (86.37%) and Alternaria alternata (98.37%) (Supplementary material Figure S2)

Mode of action of SSEO and LA combination (1:1)
The per cent reduction of ergosterol at 0.25, 0.50 and 0.75 lL/mL was found to be 11.89%, 64.86% and 95.53%, respectively (Supplementary material Figure S3, Table S5). Ergosterol is one of the principle sterol, providing rigidity and structural integrity to fungal plasma membrane. Dose dependent inhibition of ergosterol at different concentrations of SSEO and LA combination may be due to the inhibition of lanosterol 14a demethylase enzyme. Prominent leakage of vital cellular ions viz. Ca 2þ , K þ and Mg 2þ in fumigated sets of SSEO and LA combination further confirmed the plasma membrane as a probable target site of SSEO and LA action (Supplementary material Table S5). The fumigated sets also showed enhanced leakage of 260 and 280 nm absorbing materials (Supplementary material Figure S4). Excessive release of vital cellular ions and UV-absorbing materials with respect to higher doses of SSEO and LA combination is an index for membrane leakage, non-selective pore formation and damage of ion channels and transporters in plasma membrane (Helal et al. 2007).
Antiaflatoxigenic mode of action was further determined through cellular methylglyoxal (MG) assay. MG is a highly reactive cytotoxic a -oxo-aldehyde causing oxidative damage and huge production of reactive oxygen species (ROS) responsible for elevated AFB 1 secretion (Chen et al. 2004). Dose dependent inhibition of cellular MG was observed in treated sets (Supplementary material Figure S5). Concentration of MG was much higher (1097.27 lM/gFW) in control while, the combination of SSEO and LA caused significant reduction of MG at 1.0 (559.64 lM/gFW) and 2.0 lL/mL concentration (463.54 lM/gFW). Significant reduction in MG (aflatoxin inducer) production was directly correlated with AFB 1 reduction in all the treated sets of SSEO and LA combination (Das et al. 2018). Thus, the methyglyoxal inhibitory potential of combination in our study would direct to develop aflatoxin resistant herbal drugs through green transgenics.

Antioxidant activity
Highest DPPH_ and ABTS_ þ radical scavenging activity was shown in the order SSEO and LA combination (0.0024, 0.00082 mL/mL) > LA (0.0047, 0.00093 mL/mL) > SSEO (0.0051, 0.00109 mL/mL), respectively ( Figure S6a and S6b). Combined mixture of SSEO and LA showed significant IC 50 value as compared to some earlier investigated EOs viz. Satureja montana, Saturaja subspicata, and Stachys inflate essential oil. The better antioxidant potency corresponds to phenol groups in LA favoring hydrogen donation and stability of phenoxyl radicals. The high phenolic content of SSEO and LA combination perhaps caused better antioxidant activity as reported by Gholivand et al. (2010). Moreover, the high phenolic content of aromatic essential oil may cause disruption of fungal cell membrane leading to extracellular ions release as well as alteration of cellular metabolites.
2.6. In situ efficacy of SSEO and its LA combination as fumigant for stored herbal drug samples During mycoflora analysis, Picrorhiza kurroa rhizomes fumigated with SSEO and LA combination (1:1) showed pronounced efficacy against A. flavus (81.45 ± 0.58%) and other biodeteriorating fungal isolates (67.76 ± 1.82%) probably due to high phenolic content (Gholivand et al. 2010) and antioxidant nature. However, the efficacy may be further enhanced through currently used nanoencapsulation technology to manage the fungal and aflatoxin contamination of herbal drug samples.

Conclusion
The present investigation demonstrates the preservative potential of Salvia sclarea essential oil and linalyl acetate combination against common herbal drug deteriorating fungi and aflatoxin secreting fungi. Such novel combinations with strong antioxidant potential suggest their practical application as herbal drug preservative with increasing shelf life of stored herbal drugs. The methyglyoxal inhibitory action of proposed combination would be helpful to develop aflatoxin resistant herbal drug varieties through green transgenics.