Fatty acid profiling and multivariate analysis in the genus Leucas reveals its nutritional, pharmaceutical and chemotaxonomic significance

Phytochemistry 143 (2017) 72e80

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Fatty acid profiling and multivariate analysis in the genus Leucas reveals its nutritional, pharmaceutical and chemotaxonomic significance Ashish Kumar Choudhary a, P. Sunojkumar b, Girish Mishra a, * a b

Department of Botany, University of Delhi, Delhi, 110007, India Department of Botany, University of Calicut, Malappuram, Kerala, 673 635, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 April 2017 Received in revised form 13 July 2017 Accepted 21 July 2017

Genus Leucas with about 41 species found in India, is an Asian genus with separation from its close relatives in Africa based on phylogenetic evidence. Present study represents the only comprehensive phytochemical investigation on this genus. We have analyzed the seed fatty acid compositions of 26 species and five varieties of Leucas for nutritional, pharmaceutical and chemotaxonomic perspectives. The fatty acids and their composition in seeds of Leucas species, collected from different geographical regions in India, were analyzed by gas chromatography and mass spectrometry. Significant variations have been observed in fatty acid profiles among species and their varieties. We observed major fatty acids as palmitic, stearic, oleic, linoleic and laballenic acid; whereas myristic, palmitoleic, cis-vaccenic, linolenic, eicosanoic, eicosenoic, phlomic and docosanoic acid were detected in minor quantities. Laballenic and phlomic acids are unusual allenic fatty acids found in few Lamiaceae members from order Lamiales. Laballenic acid, a proven molecule of pharmaceutical importance, was observed in all the Leucas species studied. Three species of Leucas; L. helianthimifolia, L. ciliata var. vestita and L. hirta were found to contain
40% laballenic acid and can act as potential source for isolation of pharmaceutical compounds. This study also reports the presence of another allenic fatty acid, phlomic acid, in several Leucas species. Principal component analysis and hierarchical cluster analysis showed a distinct separation among the species based on abundance of similar fatty acids. The fatty acid profile appears to be overlapping at higher level and does not support separation of Asian Leucas from its African relatives and the inclusion of Asian taxa in morphologic sections. However, hierarchical clustering of L. helianthimifolia, L. ciliata var. vestita and L. hirta supported treatment under the morphologic section Astrodon. Multivariate analysis on the chemometric data also supported this cluster as the most prominent source of medicinally useful laballenic acid. Based on the FAs profile, a reconsideration of species boundaries in L. ciliata and L. marrubioides species complex is proposed. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Leucas spp.; Lamiaceae Chemotaxonomy Fatty acids Laballenic acid Phlomic acid Principal component analysis

1. Introduction Genus Leucas is a member of Lamiaceae family with about 100 species distributed in tropical to southern Africa, Arabian Peninsula, Iran to S China, Taiwan, Japan, SE Asia and up to Australia (Harley et al., 2004). Results from the recent cladistics studies (Ryding, 1998) and molecular phylogenetic analysis (Scheen and Albert, 2009) showed that Asian species form a monophyletic group whereas the Afro-Arabian species are paraphyletic. Hence,

* Corresponding author. E-mail address: [email protected] (G. Mishra). http://dx.doi.org/10.1016/j.phytochem.2017.07.007 0031-9422/© 2017 Elsevier Ltd. All rights reserved.

the genus Leucas represents only Asian species and the AfroArabian species are put under the informal name ‘African Leucas’ with a need of more studies (Scheen and Albert, 2007). Almost all the Asian Leucas are found in India and some of these species extended their distribution to Australia through South East Asia. Almost 63% of Leucas species are endemic to India (Sunojkumar and Mathew, 2009) and the diversity is seen more towards Southern parts (Singh, 2001). Various species of Leucas are widely used for traditional medicine and most species have unique medicinal properties. Phytochemical analysis of plant extracts revealed the presence of antioxidant, anti-inflammatory, analgesic, anti-diarrheal,

A.K. Choudhary et al. / Phytochemistry 143 (2017) 72e80

antimicrobial, antioxidant and insecticidal compounds (Chouhan and Singh, 2011; Kulkarni et al., 2013; Meghashri et al., 2010). A large number of phytoconstituents have been isolated from plant extract of Leucas, which include lignans, flavonoids, coumarins, steroids, terpenes, fatty acids, aliphatic long-chain compounds, coumarleucasin, leucasone, aliphatic ketols, leucasin, lactone, diterpenes, essential oil and flavone compounds (Chouhan and Singh, 2011; Al Yousuf et al., 1999; Misra et al., 1992; Hasan et al., 1991; Pradhan et al., 1990; Sadhu et al., 2006; Moody et al., 2006; Khalil et al., 1996). Silver nanoparticle synthesized from Leucas spp. extract showed effective larvicidal, pupicidal and antimicrobial activities (Antony et al., 2013; Suganya et al., 2014; Ashokkumar et al., 2014). Fatty acids (FAs) are important biomolecules that act as energy reserves, act in cell signaling and maintain membrane fluidity. Previous studies have shown that the FA composition play critical role for plants to endure various biotic and abiotic stresses, specifically chilling stress, heat stress, salt stress and wound healing (Guschina and Harwood, 2006; Nishida and Murata, 1996; Upchurch, 2008; Zhang et al., 2012; Yaeno et al., 2004; Walley et al., 2013). Seed FAs have important application in medical, chemical and food industry (Kuhnt et al., 2012; Pottel et al., 2014). FAs with unusual structures bear great demand for pharmaceutical and chemical industries as they act as precursors for various synthetic products. In addition to these economic attributes of FAs from plants, FAs have been extensively profiled for chemotaxonomic perspectives in plants (Mongrand et al., 2001, 2005; Wolff
ru-Koca et al., 2016), microet al., 2001; Dussert et al., 2008; Dog algae (Dunstan et al., 2005), fungi (Mishra et al., 2010), bacteria (Malviya et al., 2011) and cyanobacteria (Shukla et al., 2012). Until recently, approximately 2900 natural and synthetic allenic metabolites have been reported (Dembitsky and Maoka, 2007). Out of these metabolites, allenic lipids and related compounds have immense pharmaceutical potentials such as anticancer, antiinflammatory, antiviral and antibacterial activities (Dembitsky and Maoka, 2007). Lamiaceae, (especially Leucas) has been reported as a rare and unique source of unusual FAs with allenic structure (Sinha et al., 1978; Nasirullah and Osman, 1983). The laballenic acid is the first known C18 allenic FA reported from Leonotis nepetifolia (Bagby et al., 1965). Later on, this unusual FA was also reported from other species of Lamiaceae (Aitzetmüller et al., 1997). The seed oil of L. cephalotes and L. urticifolia contain 28% and 24% of laballenic acid respectively (Sinha et al., 1978; Nasirullah and Osman, 1983). Laballenic acid is an unusual FA that has not yet been reported from any other family/class except the members of Lamiaceae. Apart from laballenic acid, phlomic (20:2D7,8) and lamenallenic acid (18:3D5,6,16t) are the two other allenic FA that have also been reported from this family (Aitzetmüller et al., 1997). These two FAs are formed via chain elongation and desaturation of laballenic acid respectively. Phlomic acid and lamenallenic acid are present in Phlomis tuberosa (2.9%) and Lamium maculatum (8.8%) respectively (Aitzetmüller et al., 1997). Most unusual FAs are very important as feedstock to the oleochemical industries because of their unusual structures. Till date very few studies have been carried out on the seed oil FAs of the genus Leucas. The aim of the present study was to 1) report fatty acid profiles of 26 species and five varieties of Leucas for the first time 2) analyze inter and intraspecific variation in fatty acid content within Leucas and 3) perform multivariate analysis to establish relation between fatty acids and Leucas species to provide a chemotaxonomic perspective. 2. Results and discussion Genus Leucas are extensively exploited by the traditional healers

73

to treat various disorders, which suggest that this plant has enormous potential for the discovery of novel pharmaceutically significant compounds or drugs (Das et al., 2012). Various Leucas species are widely used as a food source and are useful to attain our nutrient requirement. This genus has nutritionally significant compounds in adequate amounts which include protein (21.3%), bcarotene, asperphenamate, phytol and phytosterol (Prakash et al., 1988; Rajyalakshmi et al., 2001; Das et al., 2012). The ethanolic extract of L. cephalotes improved the antioxidant activities through proper regulation of lipid and carbohydrate metabolism (Bavarva and Narasimhacharya, 2010). The plant extracts of various Leucas species showed CNS depressant properties, anti-inflammatory, anti-ulcer, antiepileptic, hepatoprotective, wound healing, acetylcholinesterase inhibitory, chronic central and peripheral analgesic activities (Al-Yousuf et al., 2002; Gupta et al., 2010; Ramalingam et al., 2013, Banu et al., 2012; Saha et al., 1997; Rajan and Narayan, 2012. Further, Gopi et al. (2014) and Asad et al. (2013) demonstrated that L. aspera and L. capitata has potent anti-snake venom activity, while ethyl acetate extract of L. lanata exhibited anti-parkinson and free radical scavenging activities (Ramani et al., 2013). 2.1. Collections of Leucas species Mature seeds of 26 species and five varieties of Leucas were collected from wide a range of agro-climatic zones across India (Suppl. Table 1). Detail information about sections, distribution, endemicity, locality, origin, geographical location, habitat and altitude of Leucas species are shown in suppl. Table 1. Based on morphological evidences, Asian species were treated under three infrageneric sections viz., sect. Plagiostoma, sect. Astrodon and sect. Ortholeucas (Bentham, 1830). Species collected from India represents all the three sections. Of the collected species, which includes 14 endemics species collected from Southern India. Two varieties of L. marrubioides and three varieties of L. ciliata were included to study varietal differences. Two Leucas species found in India (L. martinicensis and L. urticifolia) that comes under the African Leucas section Hemistoma are also included in the analysis (Suppl. Table 1). 2.2. Fatty acids composition Seed oil FAs from Leucas were analyzed by gas chromatographymass spectrometry. Total 13 FAs; five saturated, four monounsaturated, two polyunsaturated and two unusual FAs with allenic double bonds were observed in seed oil of Leucas species. The concentration of these FAs and prevalence of saturated, monounsaturated, polyunsaturated and unusual FAs in the investigated Leucas species are presented in Table 1. A representative GC-MS chromatogram is illustrated in Fig. 1. The composition of FAs revealed significant differences within Leucas species and their varieties. Based on concentration of FA in seed oils, five major and eight minor FAs have been identified in this present study. Out of 13 FAs, laballenic (18:2D5,6) and phlomic acid (20:2D7,8) were two unusual FAs reported in this study. The mass spectra and structures for these unusual FAs have been shown in suppl. Fig. 1. 2.2.1. Major fatty acids Palmitic, stearic, oleic, linoleic and laballenic acid were found to be the major FAs in Leucas species (Table 1). Unusual FA such as laballenic acid (C18:2D5,6) was the most prominent FA detected in Leucas species with range 13.98 ± 0.38% to 44.88 ± 0.87%, discussed in 2.2.2. Other major FAs, palmitic (C16:0), stearic (C18:0), oleic (C18:1D9) and linoleic acid (C18:2D9,12) were found to be in the range of 8.39 ± 1.26e20.55 ± 1.03%, 2.63e12.65 ± 1.55%,

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A.K. Choudhary et al. / Phytochemistry 143 (2017) 72e80

Table 1 Percentage of seed oil fatty acid compositions of collected wild Leucas species. Values are given as mole percentage. nd ¼ not detectable; SFA ¼ Saturated fatty acid; MUFA ¼ Monounsaturated fatty acid; PUFA ¼ Polyunsaturated fatty acid, UFA ¼ Unusual fatty acid. Species

C14:0

C16:0

C18:0

C20:0

C22:0

SFA

C16:1D9

C18:1D9

C18:1D11

L. angularis Benth. L. aspera (Wild). Link L. beddomei (Hook.f.) Sunojk. & P. Mathew L. biflora (Vahl) R.Br. Ex Sm L. cephalotes (Roth) Spreng L. chinensis (Retz.) R.Br. Ex Sm. L. ciliata var. vestita L. ciliata var. ciliata L. ciliata var. sericostoma L. ciliata var. new L. decemdentata (Wild.) Sm. L. eriostoma Hook. f. L. eriostoma var. lanata L. hirta (B.Heyne ex Roth) Spreng. L. helianthimifolia Desf. L. lamiifolia Desf. L. lanata var. candida L. lanceaefolia Desf. L. longifolia Benth. L. lavandulifolia Sm. L. martinicensis (Jacq.) Br. L. marrubioides var. marrubioides L. marrubioides var. pulneyensis L. nutans (Roth) spreng. L. prostrata (Hook.f.) Gamble L. rosmarinifolia Benth. L. sebaldiana Sunojk. L. sivadasaniana Sunojk. L. suffruticosa Benth. L. urticifolia (vahl) R.Br. ex Sm L. zeylanica (L.) W. T. Aiton

0.23 ± 0.01 nd 0.16

14.2 ± 0.35 16.25 ± 1.76 12.17

9.36 ± 1.48 9.92 ± 1.47 2.7

0.64 ± 0.18 0.79 ± 0.32 0.26

0.27 ± 0.12 nd 0.22

24.70 ± 1.48 26.96 ± 1.76 15.51

0.23 ± 0.11 0.19 ± 0.11 0.53

39.2 ± 0.05 33.91 ± 2.51 26.83

0.34 ± 0.15 0.07 ± 0.06 0.66

± 0.04 ± 0.04

13.33 ± 0.36 14.67 ± 0.19 11.90 ± 0.51 12.03 12.32 ± 0.73 15.51 ± 2.13 15.32 ± 1.25 14.67 ± 0.65 13.41 ± 0.07 14.57 ± 1.11 16.45 ± 0.86 12.84 ± 4.61 16.85 ± 0.55 12.60 ± 0.26 15.86 ± 3.57 14.67 ± 0.24 13.17 ± 2.51 13.20 ± 1.29 8.39 ± 1.26 16.40 ± 0.09 12.78 ± 0.39 16.71 ± 0.54 20.55 ± 1.03 16.85 ± 0.98 12.00 ± 1.52 18.46 ± 0.16 15.70 ± 1.32 18.71 ± 0.87

10.40 ± 1.12 7.44 ± 0.52 7.62 ± 1.96 2.63 6.27 ± 0.31 5.31 ± 1.19 7.89 ± 1.45 5.07 ± 0.27 6.09 ± 0.98 5.04 ± 0.90 5.78 ± 2.07 5.24 ± 0.89 7.41 ± 0.71 8.08 ± 1.16 8.85 ± 0.96 5.95 ± 0.25 8.40 ± 1.86 5.25 ± 1.11 4.45 ± 0.23 7.50 ± 0.64 5.95 ± 1.10 7.76 ± 1.29 9.77 ± 0.79 7.93 ± 0.12 11.51 ± 1.81 3.82 ± 0.55 5.33 ± 1.70 12.65 ± 1.55

0.70 0.73 1.32 0.34 0.86 0.65 0.83 0.68 1.07 0.54 0.98 3.10 0.89 0.93 1.00 0.52 0.94 0.74 0.71 1.00 0.89 0.85 1.27 1.11 1.18 0.71 nd 1.79

± 0.14 ± 0.16 ± 0.14

40.7 ± 1.54 33.72 ± 0.44 32.15 ± 0.06 16.32 24.31 ± 0.13 28.63 ± 0.12 28.15 ± 2.13 34.78 ± 0.62 30.34 ± 1.43 24.31 ± 0.51 16.91 ± 0.50 16.51 ± 1.25 27.67 ± 0.14 32.7 ± 0.75 30.52 ± 0.30 34.34 ± 0.68 31.55 ± 2.45 31.54 ± 2.03 32.33 ± 0.83 38.49 ± 0.92 34.22 ± 1.11 30.12 ± 1.03 31.19 ± 0.52 34.68 ± 0.98 26.24 ± 2.74 26.34 ± 1.05 35.12 ± 0.63 31.51 ± 1.67

nd 0.30 0.11 0.19 nd 0.64 0.32 0.95 0.99 0.78 0.61 0.25 0.69 0.37 nd 0.70 0.37 0.25 0.52 0.62 0.66 0.32 0.51 nd 0.35 1.06 0.78 0.16

0.12 0.13 nd 0.17 0.23 0.10 0.24 0.10 0.19 0.18 0.17 nd 0.47 0.13 0.30 0.10 0.08 0.16 0.19 0.19 0.15 0.38 0.38 0.17 0.08 0.13 0.18 0.19

± ± ± ± ± ± ±

0.02 0.03 0.07 0.03 0.01 0.02 0.03

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.26 0.01 0.19 0.02 0.06 0.06 0.03 0.01 0.04 0.09 0.28 0.01 0.01 0.04 0.03 0.06

16.32e40.7 ± 1.54% and 9.07 ± 0.71e35.84% of the total FA contents respectively. Monounsaturated FA mainly oleic acid (OA) predominated in all collected Leucas species and their varieties and highest percentage was observed in L. biflora (40.7 ± 1.54%). Oleic acid possesses anticancer and anti-inflammatory properties and can reduce the risk of cardiovascular diseases (Sales-Campos et al., 2013). Essential FA specifically, linoleic acid showed significant variations in FA concentration and is present in considerable amount in collected Leucas spp. with highest content in L. beddomei (35.84%). In particular, linoleic acid has pivotal role as precursors for omega-3 and eicosanoids (prostaglandins, thromboxanes and leukotrienes) biosynthesis (Youdim et al., 2000). In addition, the ratio of monounsaturated (MUFA) and polyunsaturated FAs (PUFA) is higher (
1) in majority of Leucas species studied except L. beddomei, L. ciliata var. vestita and L. eriostoma var. lanata. The highest MUFA/PUFA ratio observed is 4.57 in L. marrubioides var. marrubioides. A higher MUFA/PUFA ratio in seed oil is indicative of higher oxidative stability (Kodad and Socias, 2008). Moayedi et al. (2011) have also demonstrated oxidative stability to be directly proportional to higher MUFA/PUFA ratio in almond oil. In the present study most Leucas species have higher MUFA/PUFA ratio suggesting good seed oil stability. The presence of enormous quantity of oleic, linoleic and laballenic acid provides significant nutritional, pharmaceutical and chemotaxonomic value to Leucas seed oil. Previous studies have reported the FA contents of only two Leucas species, L. urticifolia (11.1% palmitic, 5.32% stearic, 29.7% oleic, 24.01% linoleic, 5.77% a-linolenic and 24% laballenic) and L. cephalotes (13% palmitic, 3.9% stearic, 41.6% oleic, 13.5% linoleic and 28% laballenic) (Sinha et al., 1978; Nasirullah and Osman, 1983). These results were not similar to our findings. L. ciliata var. vestita,

± 0.04 ± 0.34 ± 0.24 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.40 0.11 0.07 0.07 0.64 0.16 0.42 1.69 0.12 0.19 0.10 0.14 0.43 0.30 0.10 0.16 0.50 0.27 0.42 0.03 0.43 0.07

± 0.51

0.38 nd nd nd 0.35 0.27 0.34 nd nd 0.30 0.43 1.33 0.72 0.33 0.27 nd 0.49 0.97 0.43 0.76 nd 0.61 0.41 0.55 nd 0.81 nd 0.61

± 0.24

± 0.16 ± 0.02 ± 0.06

± ± ± ± ± ±

0.20 0.05 0.58 0.07 0.05 0.14

± ± ± ±

0.26 0.39 0.12 0.26

± 0.24 ± 0.15 ± 0.10 ± 0.15 ± 0.25

24.93 22.97 20.84 15.17 20.03 21.84 24.62 20.52 20.76 20.63 23.81 22.51 26.34 21.06 26.28 21.24 23.08 20.32 14.17 25.85 19.77 26.31 32.38 26.61 24.77 23.93 21.21 33.95

± 1.12 ± 0.52 ± 1.96 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.73 2.13 1.45 0.65 0.98 1.11 2.07 4.61 0.71 1.16 3.57 0.25 2.51 1.29 1.26 0.64 1.10 1.29 1.03 0.98 1.81 0.55 1.70 1.55

0.21 0.29 0.22 0.34 0.17 0.34 1.84 0.16 0.39 0.15 0.39 0.28 0.31 0.29 0.30 0.12 0.13 0.16 0.08 nd 0.20 0.17 0.24 0.22 0.20 0.17 nd 0.16

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.09 0.28 0.92 0.08 0.19 0.09 0.27 0.15 0.18 0.15 0.16 0.06 0.07 0.09 0.03

± ± ± ± ± ±

0.11 0.09 0.17 0.11 0.10 0.09

± 0.08

± 0.08 ± 0.07

± ± ± ± ± ± ± ± ±

0.09 0.43 0.12 0.14 0.01 0.01 0.12 0.30 0.53

± ± ± ± ± ± ± ±

0.19 0.11 0.02 0.12 0.36 0.07 0.13 0.15

± ± ± ±

0.13 0.02 0.38 0.09

L. ciliata var. ciliate, L. ciliata var. sericostoma, L. ciliata var. new, L. marrubioides var. marrubioides and L. marrubioides var. pulneyensis are varieties of three different Leucas spp. and collected from different ecological habitats. Within Leucas varieties, significant variations were also observed in major and minor FAs composition (Table 1). The factors discussed in section 2.2.4 might be responsible for this incongruence. 2.2.2. Unusual fatty acids Presence of unusual allenic FA, laballenic acid was observed in all collected species (Table 1). Laballenic acid was prominent in all species with highest concentration in L. helianthimifolia (44.88 ± 0.87%). A large variation was observed in laballenic acid concentration among Leucas species. The seed oil of L. cephalotes and L. urticifolia have been already investigated and they contain laballenic acid as major FA with 28% and 24% of total FA contents respectively (Sinha et al., 1978). Laballenic acid, first known unusual FA is proven to have anti-inflammatory properties (Patel et al., 2015). This FA significantly reduces production of lipopolysaccharides (LPS) induced tumor necrosis factor (TNF)-a and interleukin (IL)-1b in rats (Patel et al., 2015). Hence, such novel therapeutic role of laballenic acid can be utilized for the development of antiinflammatory drugs. Phlomic acid (20:2D7,8), another allenic FA had been identified and reported in genus Leucas for the first time in this study. In our study minor quantities of phlomic acid was observed in 11 species ranging from 0.22 ± 0.01% to 1.86 ± 0.05% with highest in L. ciliata var. new (1.86 ± 0.05%). Previously, Phlomic acid has been reported in several other members of Lamiaceae with highest concentration in Phlomis tuberosa (2.9%) (Aitzetmüller et al., 1997). Lamiaceae comprises of 236 genera and out of them

A.K. Choudhary et al. / Phytochemistry 143 (2017) 72e80

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C20:1D11

MUFA

C18:2D9,12

C18:3D9,12,15

PUFA

MUFA/PUFA

C18:2D5,6

C20:2D7,8

UFA

0.86 ± 0.37 nd 0.73

40.63 ± 0.37 34.17 ± 2.51 28.75

16.35 ± 1.73 23.22 ± 1.03 35.84

0.60 ± 0.02 0.42 ± 0.15 0.79

16.95 ± 1.73 23.64 ± 1.03 36.63

2.39 1.44 0.78

17.60 ± 0.00 15.16 ± 1.62 19.06

nd nd nd

17.6 ± 0.00 15.16 ± 1.62 19.06

1.41 ± 0.82 0.71 ± 0.33 0.94 ± 0.39 0.86 1.07 ± 0.29 1.7 ± 0.41 2.34 ± 0.08 0.31 ± 0.04 1.38 ± 0.76 1.20 ± 0.31 2.88 ± 0.46 1.42 ± 0.18 1.22 ± 0.65 0.88 ± 0.43 2.31 ± 0.41 0.25 ± 0.02 1.02 ± 0.62 0.56 ± 0.22 2.53 ± 0.63 2.39 ± 0.44 1.09 ± 0.56 1.16 ± 0.25 1.73 ± 0.44 1.11 ± 0.23 0.67 ± 0.03 1.21 ± 0.21 nd 0.82 ± 0.31

42.32 ± 1.54 35.02 ± 0.44 33.42 ± 0.39 17.71 25.55 ± 0.29 31.31 ± 0.41 32.65 ± 2.13 36.20 ± 0.62 33.10 ± 1.43 26.44 ± 0.51 20.79 ± 0.50 18.46 ± 1.25 29.89 ± 0.65 34.24 ± 0.75 33.13 ± 0.41 35.41 ± 0.68 33.07 ± 2.45 32.51 ± 2.03 35.46 ± 0.83 41.5 ± 0.92 36.17 ± 1.11 31.77 ± 1.03 33.67 ± 0.52 36.01 ± 0.98 27.46 ± 2.74 28.78 ± 1.05 35.9 ± 0.63 32.65 ± 1.67

15.10 ± 0.53 25.00 ± 1.97 22.51 ± 3.10 23.19 23.38 ± 0.40 29.26 ± 2.01 25.49 ± 0.95 24.44 ± 0.58 29.40 ± 0.93 29.65 ± 1.13 13.88 ± 4.06 13.26 ± 2.88 24.67 ± 1.65 26.63 ± 0.52 23.65 ± 0.73 27.80 ± 0.28 26.48 ± 1.58 23.50 ± 0.85 21.45 ± 2.12 9.07 ± 0.71 22.61 ± 0.89 19.25 ± 0.64 15.28 ± 0.19 15.99 ± 1.42 23.54 ± 2.06 25.57 ± 0.22 24.63 ± 1.40 17.72 ± 0.34

0.45 ± 0.12 0.47 ± 0.09 0.94 ± 0.42 0.88 1.14 ± 0.05 1.07 ± 0.15 1.28 ± 0.15 0.63 ± 0.05 1.05 ± 0.07 1.10 ± 0.17 0.71 ± 0.12 0.81 ± 0.18 0.91 ± 0.20 0.68 ± 0.12 0.83 ± 0.22 0.57 ± 0.02 1.17 ± 0.61 4.31 ± 0.23 nd nd 0.61 ± 0.15 1.38 ± 0.23 0.62 ± 0.16 0.7 ± 0.08 0.69 ± 0.21 1.06 ± 0.15 1.27 ± 0.18 0.51 ± 0.14

15.55 ± 0.53 25.47 ± 1.97 23.45 ± 3.10 24.07 24.52 ± 0.40 30.33 ± 2.01 26.77 ± 0.95 25.07 ± 0.58 30.55 ± 0.93 30.75 ± 1.13 14.59 ± 4.06 14.07 ± 2.88 25.58 ± 1.65 27.31 ± 0.52 24.48 ± 0.73 28.37 ± 0.28 27.65 ± 1.58 27.81 ± 0.85 21.45 ± 2.12 9.07 ± 0.71 23.22 ± 0.89 20.63 ± 0.64 15.90 ± 0.19 16.69 ± 1.42 24.23 ± 2.06 26.63 ± 0.22 25.90 ± 1.40 18.23 ± 0.34

2.72 1.37 1.42 0.73 1.04 1.03 1.21 1.44 1.08 0.85 1.42 1.31 1.16 1.25 1.35 1.24 1.19 1.16 1.65 4.57 1.55 1.53 2.11 2.15 1.13 1.08 1.38 1.79

16.82 16.45 22.21 43.04 29.57 14.69 13.98 18.12 15.53 22.16 40.31 44.88 18.09 16.04 15.68 14.90 16.09 18.99 28.84 22.92 20.75 20.88 17.99 20.14 22.24 20.20 16.93 15.06

laballenic acid has been reported from 13 genera (Aitzetmüller et al., 1997). The presence or absence of these unusual FAs in seed oil of Lamiaceae family members could be useful for evolutionary and chemotaxonomic studies (Aitzetmüller et al., 1997). Furthermore, previous reports suggested that combining information from the relative abundance of usual and unusual FAs is effective for  and Velasco, 2000; Ozcan, taxonomic purpose (Pujadas-Salva 2013). The significant variation in seed oil FAs composition forms the basis of selection of specific plant genus/species for chemotaxonomic, pharmaceutical, nutritional and industrial usages. 2.2.3. Minor fatty acids In the seed oil of Leucas species; eight minor FAs were observed (Table 1). Myristic (C14:0) palmitoleic (C16:1D9), cis-vaccenoic (C18:1D11), linolenic (C18:3D9,12,15), eicosanoic (C20:0), eicosenoic (C20:1D11), phlomic (C20:2D7,8) and docosanoic acid (C22:0) were estimated to be in the range of 0.08 ± 0.01e0.47 ± 0.26%, 0.08 ± 0.03e1.84 ± 0.92%, 0.07 ± 0.06e1.06 ± 0.02%, 0.42 ± 0.15e4.31 ± 0.23%, 0.26e3.10 ± 1.69%, 0.25 ± 0.02e2.88 ± 0.46%, 0.22 ± 0.01e1.86 ± 0.05% and 0.22e1.33 ± 0.58% of the total FAs. Among the minor FAs, eicosenoic acid predominated in L. hirta (2.88 ± 0.46%), L. marrubioides var. marrubioides (2.53 ± 0.63%), L marrubioides var. pulneyensis (2.39 ± 0.44%) and L. lanceaefolia (2.31 ± 0.41%). Linolenic acid (Omega-3) is an essential FA; ten species have more than 1% alinolenic acid out of total FA contents, highest concentration in L. martinicensis (4.31 ± 0.23%). Earlier studies showed L. urticifolia has 5.77% linolenic acid but only 1.27 ± 0.18% a-linolenic acid was observed in our study. Phlomic acid, an unusual FA was first time observed in Leucas species, discussed in section 2.2.2. These minor FAs have not been obtained in all the species. C14:0, C16:1D9,

± 0.62 ± 0.19 ± 1.06 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.15 1.58 0.38 0.20 0.27 1.85 0.28 0.87 1.27 0.17 0.79 1.06 0.16 2.04 0.50 1.26 0.66 0.15 0.63 1.83 0.29 2.36 2.86 0.44

0.28 nd nd nd 0.26 1.73 1.86 nd nd nd 0.42 nd nd 0.23 0.35 nd nd nd nd 0.46 nd 0.36 nd 0.32 nd 0.22 nd nd

± 0.14

± 0.09 ± 0.14 ± 0.05

± 0.13

± 0.81 ± 0.14

± 0.19 ± 0.15 ± 0.05 ± 0.01

17.10 ± 0.62 16.45 ± 0.19 22.21 ± 1.06 43.04 29.83 ± 0.15 16.42 ± 1.58 15.84 ± 0.38 18.12 ± 0.20 15.53 ± 0.27 22.16 ± 1.85 40.73 ± 0.28 44.88 ± 0.87 18.09 ± 1.27 16.27 ± 0.81 16.03 ± 0.79 14.9 ± 1.06 16.09 ± 0.16 18.99 ± 2.04 28.84 ± 0.50 23.38 ± 1.26 20.75 ± 0.66 21.24 ± 0.15 17.99 ± 0.63 20.46 ± 1.83 22.24 ± 0.29 20.42 ± 2.36 16.93 ± 2.86 15.06 ± 0.44

C18:1D11, C20:0, C20:1D11, C20:2D7,8 and C22:0 were not reported in previous studies (Sinha et al., 1978; Nasirullah and Osman, 1983). 2.2.4. Factors affecting fatty acids composition Significant variations in seed oil FAs composition amongst Leucas species and varieties were observed (Table 1). Some of the collected Leucas species are endemic to particular habitat and it suggests that these species are acclimated to specific climatic condition (Suppl. Table 1). In several plant species, FA contents can be modulated by their geographical location and climatic conditions (Johansson et al., 2000). We observed significant differences in major and minor FA concentrations (Table 1) within varieties of L. ciliata, L. eriostoma and L. marrubioides. There are several factors affecting seed oil FA contents within a species. Gulfraz et al. (2009) noted variation in FA contents within same variety of olive. The quantitative variation of seed oil FA contents is directly influenced by environment and habitat (Linder, 2000). Even nutrient availability, latitude, salinity, moisture, and average day light may also have significant impact on FA content (Angelini et al., 1997; Ghebretinsae et al., 2008; Wu et al., 1998; Hrastar et al., 2012). Fatty acid composition of Soybean seed oil is reported to be affected by various environmental factors including temperature, drought stress, planting site, planting day, overall rainfall, seed-fill period, average solar radiation, photoperiod, light quality and intensity, soil type, irrigation and nutrition (Hou et al., 2006). Graham et al. (2016) have reported unexpected significant variation in predominated unusual medium chain FAs (lauric acid, 12:0; myristic acid, 14:0) and regular FAs (oleic acid, 18:1D9; linoleic acid, 18:2D9,12) in Cuphea species due to adaptive radiation, speciation and habitat conditions. The reason of the significant variation in FA composition among some of our Leucas species with those reported earlier

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Fig. 1. Representative GC chromatogram of fatty acid methyl esters (FAMEs) of Leucas species. [peak 1, myristic acid (14:0); 2, palmitic acid (16:0); 3, palmitoleic acid (16:1D9); 4, stearic acid (18:0), 5, oleic acid (18:1D9), 6, cis-vaccenoic acid (18:1D11), 7, linoleic acid (18:2D9,12) 8, laballenic acid (18:2D5,6) 9, linolenic acid(18:3D9,12,15), 10, eicosanoic acid (20:0); 11, eicosenoic acid (20:1D11), 12, phlomic acid (20:2D7,8), 13, docosanoic acid (22:0)].

might be due to the variation of ecological habitats, geographical locations and diverse climatic conditions. Though it has been reported that geographical locations affect the FA profiles of membranes, changes observed in seed oil FAs could also be under genetic control (O'Neill et al., 2003).

acids while high negative loading factor for laballenic acid only. These results show that palmitic or stearic acid might be a precursor or limiting factor for laballenic acid biosynthesis. Briefly, PCA grouped all collected Leucas species on the basis of abundance of particular FAs. PCA was performed to gain better understanding of the relation between Leucas species and their FA variables.

2.3. Multivariate analysis 2.4. Chemotaxonomy Principal component analysis (PCA) was carried out for total 13 FAs to test their relationship among the different Leucas species. All 13 FAs as variables were further analyzed using PCA software. PCA is a statistical analytical method which reduces the dimensionality of original data set and identifies new variables (Principles comr, 2008). The observations were plotted on a biponents) (Ringne plot graph which demonstrated the correlation between each FA variable with Leucas species. The Pearson correlation data matrix of FA variables was used for bi-plot graph. Eigenvalues are indicators of relative importance of each dimension. The first five PCs had 2.859, 2.377, 2.145, 1.452 and 1.274 eigenvalues respectively (Suppl. Table 2). The first PC explained 21.99% and second PC accounted 18.28% of total variations (Fig. 2). Other three PCs explained 16.49%, 11.16% and 9.79% of total variations respectively. The PC1- PC2 and PC1-PC3 score plots accounted for 40.28% and 38.49% of total variance respectively (Fig. 2). The PC1-PC2 score plot shows that all FA vectors were distributed within the correlation circle (Fig. 2A). PC1 elucidated high positive loading factor for C18:2D5,6 (0.618), C20:0 (0.753), C20:D11 (0.632), C22:0 (0.714) and negative high loading factor for only linoleic acid (C18:2D9,12; 0.731) as shown in Fig. 2A (Suppl. Table 2). PC2 showed positive correlation with high loading factor for C16:0 (0.546), C18:0 (0.859) and C18:1D9 (0.719), while negative correlation and high loading factor for predominated laballenic acid (C18:2D5,6; 0.639) (Fig. 2A). In addition, PC3 contributes only high positive loading factor for minor FAs, phlomic (C20:2D7,8, 0.815) and palmitoleic acid (C16:1D9; 0.775) (Fig. 2B). PC1-PC2 score plot and hierarchical clustering analysis (HCA) revealed that L. helianthimifolia, L. ciliata var. vestita and L. hirta grouped together on the basis of higher laballenic acid concentration among all collected species (Fig. 3). These Leucas species are new source of laballenic acid, it bears pharmaceutical potential that can be harvested for future drugs against inflammatory diseases and might be useful as precursor of other therapeutic agents. The biosynthetic pathway of laballenic acid is not reported yet. The PC2 showed high positive loading factor for two saturated FAs, palmitic and stearic

Algorithmic hierarchical clustering (AHC) dendrogram generated by using euclidian distance and ward method has been shown in Fig. 4. The dendrogram is divided into four major subgroups on the basis of similarity of FAs: the first three groups represent Leucas species grouped on the basis of abundance of major usual FAs, while second group contains Leucas species having higher unusual laballenic acids (more than 40%). The clustering differentiated by HCA also unveils chemical similarities and differences that are not seen otherwise (Custodio et al., 2003). The FA profiles do not provide a clear cut evidence to support separation of Asian Leucas from its African relatives as suggested by Scheen and Albert (2007, 2009). Similarly, at infrageneric level, Leucas species did not show meaningful clusters. However, PCA and HCA suggested that L. helianthimifolia, L. ciliata var. vestita and L. hirta grouped together and possess more than 40% laballenic acid (Figs. 3 and 4). These taxa are more or less endemic to Western Ghats in South India, possess similar morphological characters and were included in the morphologic section Astrodon by classical taxonomists (Bentham, 1830). The ongoing molecular phylogenetic studies by the authors also supported this cluster as well as the close relationships among certain species (L. aspera and L. lavandulifolia; L. angularis and L. biflora). The FA profiles suggest a taxonomic reconsideration of relationship in the L. ciliata and L. marrubioides species complexes as the infrageneric components falls wide apart in the dendrogram. The less-resolved cluster at infra-generic level, based on FAs profile, could be because of the recent radiation of this genus in India and inadequate expansion of morphospecies. 3. Conclusion Inadequate investigation has been done on genus Leucas seed oil FAs. The present study provides the first comprehensive analyses of FA composition of 31 Leucas species and varieties mostly endemic to Indian subcontinent. Large variations in concentration of palmitic, stearic, oleic, linoleic and laballenic acids were observed. The

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Fig. 2. Loading plot for principal component analysis of 13 fatty acids from collected Leucas species (A) PC1-PC2 and (B) PC1-PC3.

Fig. 3. Scatter biplot diagrams of seed oil fatty acid profiles and Leucas species according to principal component 1(PC1) and principal component 2 (PC2) axes of analysis. Three Leucas species with
40% laballenic acid were grouped together and shown in circle.

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Fig. 4. Dendrogram obtained by hierarchical clustering analysis of collected Leucas species and varieties.

significant variation in usual and unusual FAs of Leucas seed oil might be due to causes like collection from wide range of ecological habitats, local climatic condition, region specific abiotic stresses and migrated species. All collected species show high content of nutritionally important unsaturated FAs (especially, oleic and linoleic acid) and pharmaceutically important unusual FA especially, laballenic acid. High laballenic acid containing Leucas species such as L. helianthimifolia, L. ciliata var. vestita and L. hirta bears great potential to be explored for pharmaceutical and commercial purposes. Previous report showed that seeds of Leucas contain 24e28.5% oil of total weight (Aitzetmüller et al., 1997). Due to the presence of higher unsaturated (oleic and linoleic), unusual (laballenic) FA and oil content in seeds, Leucas is a unique study system for the FA metabolism and biosynthesis of allenic FAs. Moreover, FA profiles generated by us were also found to be useful for chemotaxonomic

studies. In current analyses, many species were clustered together as seen in the molecular phylogeny of Leucas. All collected species show high content of nutritionally important unsaturated FAs (especially, oleic and linoleic acid) and pharmaceutically important unusual FA especially, laballenic acid. Aitzetmüller et al. (1997) suggested that presence or absence of seed oil unusual FAs in Lamiaceae could be an important indicator of chemotaxonomy. The unusual laballenic and phlomic FAs can act as markers for further division of Lamiaceae and for better understanding the evolution and biochemistry of FAs in the group. The present investigation revealed the importance of seed oil FAs in chemotaxonomical studies of genus Leucas. In future, a comprehensive molecular systematic study of more species and related genera with FA profile across continents would help in better understanding of the evolution of the genus Leucas.

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4. Materials and methods

References

4.1. Chemicals and reagents

Aitzetmüller, K., Tsevegsüren, N., Vosmann, K., 1997. A new allenic fatty acid in Phlomis (Lamiaceae) seed oil. Lipid-Fett 99, 74e78. Al-Yousuf, M.H., Ali, B.H., Bashir, A.K., Tanira, M.O.M., Blunden, G., 2002. Central nervous system activity of Leucas inflata Benth. in mice. Phytomedicine 9 (6), 501e507. Al Yousuf, M.H., Bashir, A.K., Blunden, G., Yang, M.H., Patel, A.V., 1999. Coumarleucasin and leucasone from Leucas inflata roots. Phytochemistry 51, 95e98. Angelini, L.G., Moscheni, E., Colonna, G., Belloni, P., Bonari, E., 1997. Variation in agronomic characteristics and seed oil composition of new oilseed crops in central Italy. Ind. Crops Prod. 6, 313e323. Antony, J.J., Nivedheetha, M., Siva, D., Pradeepha, G., Kokilavani, P., Kalaiselvi, S., Sankarganesh, A., Balasundaram, A., Masilamani, V., Achiraman, S., 2013. Antimicrobial activity of Leucas aspera engineered silver nanoparticles against Aeromonas hydrophila in infected Catla catla. Colloid Surf. B 109, 20e24. Asad, M.H.H.B., Razi, M.T., Durr-e-Sabih, Najamus-Saqib, Q., Nasim, S.J., Murtaza, G., Hussain, I., 2013. Anti-venom potential of Pakistani medicinal plants: inhibition of anticoagulation activity of Naja naja karachiensis toxin. Curr. Sci. 105 (10), 1419e1424. Ashokkumar, S., Ravi, S., Kathiravan, V., Velmurugan, S., 2014. Rapid biological synthesis of silver nanoparticles using Leucas martinicensis leaf extract for catalytic and antibacterial activity. Environ. Sci. Pollut. Res. 21, 11439e11446. Bagby, M.O., Smith Jr., C.R., Wolff, I.A., 1965. Laballenic acid, A new allenic acid from Leonotis nepetaefolia seed oil. J. Org. Chem. 30, 4227e4229. Bentham, G., 1830. In: Wallich, N. (Ed.). Plantae Asiaticae Rariorum I, London, pp. 60e62. Banu, S., Bhaskar, B., Balasekar, P., 2012. Hepatoprotective and antioxidant activity of Leucas aspera against D-galactosamine induced liver damage in rats. Pharm. Biol. 50 (12), 1592e1595. Bavarva, J.H., Narasimhacharya, A.V.R.L., 2010. Leucas cephalotes regulates carbohydrate and lipid metabolism and improves antioxidant status in IDDM and NIDDM rats. J. Ethnopharmacol. 127 (1), 98e102. Chouhan, H.S., Singh, S.K., 2011. A review of plants of genus Leucas. J. Pharmacogn. Phytother. 3, 13e26. Christie, W.W., 1993. Preparation of ester derivatives of fatty acids for chromatographic analysis. In: Advances in Lipid Methodology Two. Oily Press, Dundee, Scotland, pp. 69e111. Custodio, A.R., Ferreira, M., Negri, G., Salatino, A., 2003. Clustering of comb and propolis waxes based on the distribution of aliphatic constituents. J. Braz. Chem. Soc. 14, 354e357. Das, S.N., Patro, V.J., Dinda, S.C., 2012. A review: Ethnobotanical survey of genus Leucas. Pharmacogn. Rev. 6 (12), 100e106. Dembitsky, V.M., Maoka, T., 2007. Allenic and cumulenic lipids. Prog. Lipid Res. 46, 328e375. €
ru-Koca, A., Ozcan, Dog T., Yıldırımlı, S¸., 2016. Chemotaxonomic perspectives of the Paracaryum (Cynoglosseae, Boraginaceae) taxa based on fruit fatty acid composition. Phytochemistry 131, 100e106. Dunstan, G.A., Brown, M.R., Volkman, J.K., 2005. Cryptophyceae and Rhodophyceae; chemotaxonomy, phylogeny, and application. Phytochemistry 66, 2557e2570. €t, T., 2008. Effectiveness of the fatty acid Dussert, S., Laffargue, A., de Kochko, A., Joe and sterol composition of seeds for the chemotaxonomy of Coffea subgenus Coffea. Phytochemistry 69, 2950e2960. Ghebretinsae, A.G., Graham, S.A., Camilo, G.R., Barber, J.C., 2008. Natural infraspecific variation in fatty acid composition of Cuphea (Lythraceae) seed oils. Ind. Crops Prod. 27, 279e287. Gopi, K., Renu, K., Jayaraman, G., 2014. Inhibition of Naja naja venom enzymes by the methanolic extract of Leucas aspera and its chemical profile by GCeMS. Toxicol. Rep. 1, 667e673. , G.P.C., Murad, A.M., Rech, E.L., Cavalcanti, T.B., Inglis, P.W., 2016. Graham, S.A., Jose Patterns of fatty acid composition in seed oils of Cuphea, with new records from Brazil and Mexico. Ind. Crops Prod. 87, 379e391. Gulfraz, M., Kasuar, R., Arshad, G., Mehmood, S., Minhas, N., Asad, M.J., Ahmad, A., Siddique, F., 2009. Isolation and characterization of edible oil from wild olive. Afr. J. Biotechnol. 8, 3734e3738. Gupta, J.K., Upmanyu, N., Patnaik, A.K., Mazumder, P.M., 2010. Evaluation of Antiulcer activity of Leucas lavandulifolia on mucosal lesion in rat. Asian J.Pharm. Clin. Res. 3 (2), 118e120. Guschina, I.A., Harwood, J.L., 2006. Mechanisms of temperature adaptation in poikilotherms. FEBS Lett. 580, 5477e5483. Hasan, M., Burdi, D.K., Ahmad, V.U., 1991. Leucasin, a triterpene saponin from Leucas nutans. Phytochemistry 30, 4181e4183. Harley, R.M., Atkins, S., Budantsev, A.L., Cantino, P.D., Conn, B.J., Greyer, R., Harley, M.M., De Kok, R., Krestovskaja, T., Morales, R., Paton, A.J., Ryding, O., Upson, T., 2004. Labiatae. In: Kubitzki, K., Kadereit, J.W. (Eds.), The Families and Genera of Vascular Plants 7. Springer, Berlin, pp. 167e275. Hou, G., Ablett, G.R., Pauls, K.P., Rajcan, I., 2006. Environmental effects on fatty acid levels in soybean seed oil. J. Am. Oil Chem. Soc. 83 (9), 759e763. Hrastar, R., Abramovic, H., Kosir, I.J., 2012. In situ quality evaluation of Camelina sativa landrace. Eur. J. Lipid Sci. Technol. 114, 343e351. Johansson, A., Laine, T., Linna, M.M., Kallio, H., 2000. Variability in oil content and fatty acid composition in wild northern currants. Eur. Food Res. Technol. 211, 277e283. Khalil, A.T., Gedara, S.R., Lahloub, M.F., Halim, A.F., 1996. Diterpenes and a flavone

Analytical grade chemicals and GC grade solvents were used for all analyses. The chemicals and glass/plastic ware used were, sodium chloride (Merck), methanol (Sigma), hexane (Sigma), conical glass tube (Pyrex), teflon coated cap (Pyrex), glass pasteur pipette (Fisher scientific) and nitrogen gas. 4.2. Samples collection Dried, mature seeds of Leucas were collected from Western Ghats, Kerala, Tamil Nadu, Andhra Pradesh, Rajasthan, Maharashtra, Assam, Bihar and Uttar Pradesh, India. Samples were collected from wild populations located in different climatic conditions (Suppl. Table 1). Healthy, mature and dry seeds were separated, cleaned and stored for analyses. 4.3. Fatty acid methyl ester analyses Fatty acid methyl esters were prepared using method reported by Christie (1993) with minor modifications. Ten seeds were crushed using mortar-pestle and transferred to teflon lined screw capped glass tube to which 1 ml of 2% (w/v) HCl in methanol was added and the solution was incubated at 80  C for 1 h. FAMEs were extracted twice by adding 1 ml of 0.9% NaCl and 2 ml of hexane, vortexing for 40 s and centrifuged for phase separation. The collected hexane layer was dried under stream of nitrogen gas and resuspended in 100 ml of hexane for GC-MS analysis. The fatty acid methyl esters were analyzed using gas chromatography-mass spectrometer (5977A MSD coupled with 7890B GC series, Agilent Technologies) equipped with a 30 m  0.25 mm x 0.25 mm DB-wax capillary column (Agilent Technologies). The oven temperature was raised from 50  C to 230  C at a rate of 5  C min1 with carrier gas flow at 1.8 ml/min. The injection volume was kept at 1 ml with split ratio of 20:1. FAMEs were identified through comparison of mass spectral data to NIST library. 4.4. Statistical analysis The statistical analyses were performed to study the relationship within Leucas on the basis of their seed fatty acid composition. Principle component analysis (PCA) and hierarchical clustering analysis (HCA) was conducted with euclidean distance similarity index of fatty acid composition using XLSTAT software. PCA is a multivariate statistical technique that generates new set of orthogonal axes (or variable) known as principal components from original data set. The eigenvalues and coefficient loading matrices were also obtained from PCA. Acknowledgements GM and AKC are grateful to University of Delhi for providing the Research and Development grant (RC/2014). PS is grateful to the Department of Biotechnology (DBT), Government of India for the Leucas phylogeny project grant support (BT/PR5423/BCE/8/907/ 2012). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2017.07.007.

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from Leucas neufliseana. Phytochemistry 41, 1569e1571. Kodad, O., Socias, I.C.R., 2008. Variability of oil content and of major fatty acid composition in almond (Prunus amygdalus Batsch) and its relationship with kernel quality. J. Agric. Food. Chem. 56, 4096e4101. Kuhnt, K., Degen, C., Jaudszus, A., Jahreis, G., 2012. Searching for health beneficial n3 and n-6 fatty acids in plant seeds. Eur. J. lipid Sci. Technol. 114, 153e160. Kulkarni, R.R., Shurpali, K., Puranik, V.G., Sarkar, D., Joshi, S.P., 2013. Antimycobacterial labdane diterpenes from Leucas stelligera. J. Nat. Prod. 76, 1836e1841. Linder, C.R., 2000. Adaptive evolution of seed oils in plants: accounting for the biogeographic distribution of saturated and unsaturated fatty acids in seed oils. Am. Nat. 156, 442e458. Malviya, N., Yadav, A.K., Yandigeri, M.S., Arora, D.K., 2011. Diversity of culturable Streptomycetes from wheat cropping system of fertile regions of Indo-Gangetic Plains, India. World J. Microbiol. Biotechnol. 27, 1593e1602. Meghashri, S., Kumar, H.V., Gopal, S., 2010. Antioxidant properties of a novel flavonoid from leaves of Leucas aspera. Food Chem. 122, 105e110. Mishra, A.K., Singh, A., Singh, S.S., 2010. Diversity of Frankia strains nodulating € HippOphae salicifolia D. Don using FAME profiling as Chemotaxonomic markers. J. Basic Microb. 50, 318e324. Misra, T.N., Singh, R.S., Prasad, C., Singh, S., 1992. Two aliphatic ketols from Leucas aspera. Phytochemistry 32, 199e201. Moayedi, A., Rezaei, K., Moini, S., Keshavarz, B., 2011. Chemical composition of oils from several wild almond species. J. Am. Oil Chem. Soc. 88, 503e508. Mongrand, S., Badoc, A., Patouille, B., Lacomblez, C., Chavent, M., Cassagne, C., Bessoule, J.J., 2001. Taxonomy of gymnospermae: multivariate analyses of leaf fatty acid composition. Phytochemistry 58, 101e115. Mongrand, S., Badoc, A., Patouille, B., Lacomblez, C., Chavent, M., Bessoule, J.J., 2005. Chemotaxonomy of the Rubiaceae family based on leaf fatty acid composition. Phytochemistry 66, 549e559. Moody, J.O., Gundidza, M., Wyllie, G., 2006. Essential oil composition of Leucas milanjiana Guerke. Flavour. Frag. J. 21, 872e874. Nasirullah, A.F., Osman, S.M., 1983. Leucas urticaefolia seed oil-a rich source of laballenic acid. Fette, seifen. Anstrichmittel 85, 314e315. Nishida, I., Murata, N., 1996. Chilling sensitivity in plants and cyanobacteria: the crucial contribution of membrane lipids. Ann. Rev. Plant Biol. 47, 541e568. O'Neill, C.M., Gill, S., Hobbs, D., Morgan, C., Bancroft, I., 2003. Natural variation for seed oil composition in Arabidopsis thaliana. Phytochemistry 64, 1077e1090. Ozcan, T., 2013. Molecular (RAPDs and Fatty acid) and micromorphological variations of Echium italicum L. populations from Turkey. Plant Syst. Evol. 299, 631e641. Patel, N.K., Khan, M.S., Bhutani, K.K., 2015. Investigations on Leucas cephalotes (Roth.) Spreng. for inhibition of LPS-induced pro-inflammatory mediators in murine macrophages and in rat model. EXCLI J. 14, 508e516. Pottel, L., Lycke, M., Boterberg, T., Foubert, I., Pottel, H., Duprez, F.R., Goethals, L., Debruyne, P.R., 2014. Omega-3 fatty acids: physiology, biological sources and potential applications in supportive cancer care. Phytochem. Rev. 13, 223e244. Prakash, D., Jain, R.K., Misra, P.S., 1988. Amino acid profiles of some under-utilised seeds. Plant Foods Hum. Nutr. 38, 235e241. Pradhan, B.P., Chakraborty, D.K., Subba, G.C., 1990. A triterpenoid lactone from Leucas aspera. Phytochemistry 29, 1693e1695. , A.J., Velasco, L., 2000. Comparative studies on Orobanche cernua L. Pujadas-Salva and Orobanche cumana Wallr. (Orobanchaceae) in the Iberian Peninsula. Bot. J. Linn. Soc. 134, 513e527. Rajan, C., Narayan, R.S., 2012. Chronic central and peripheral analgesic activity of

Ethanolic Extract of the leaves of Leucas indica (EELI) in rodent models. Drug Inven. Today 4 (8), 11e13. Rajyalakshmi, P., Venkatalaxmi, K., Venkatalakshmamma, K., Jyothsna, Y., Devi, K.B., Suneetha, V., 2001. Total carotenoid and beta-carotene contents of forest green leafy vegetables consumed by tribals of South India. Plant Foods Hum. Nutr. 56, 225e238. Ramalingam, R., Nath, A.R., Madhavi, B.B., Nagulu, M., Balasubramaniam, A., 2013. Free radical scavenging and antiepileptic activity of Leucas lanata. J. Pharm. Res. 6 (3), 368e372. Ramani, R., Anisetti, R.N., Boddupalli, B.M., Malothu, N., Arumugam, B.S., 2013. Antiparkinson's and free radical scavenging study of ethyl acetate fraction of ethanolic extract of Leucas lanata. Drug Invent. Today 5 (3), 251e255. r, M., 2008. What is principal component analysis? Nat. Biotechnol. 26, Ringne 303e304. Ryding, O., 1998. Phylogeny of the Leucas group (Lamiaceae). Syst. Bot. 23, 235e247. Sadhu, S.K., Okuyama, E., Fujimoto, H., Ishibashi, M., 2006. Diterpenes from Leucas aspera inhibiting prostaglandin-induced contractions. J. Nat. Prod. 69, 988e994. Sales-Campos, H., Souza, P.R., Peghini, B.C., da Silva, J.S., Cardoso, C.R., 2013. An overview of the modulatory effects of oleic acid in health and disease. Mini Rev. Med. Chem. 13, 201e210. Saha, K., Mukherjee, P.K., Das, J., Pal, M., Saha, B.P., 1997. Wound healing activity of Leucas lavandulaefolia Rees. J. Ethnopharmacol. 56 (2), 139e144. Scheen, A.C., Albert, V.A., 2007. Nomenclatural and taxonomic changes within the Leucas clade (Lamioideae: Lamiaceae). Syst. Geogr. 77, 229e238. Scheen, A.C., Albert, V.A., 2009. Molecular phylogenetics of the Leucas group (Lamioideae; Lamiaceae). Syst. Bot. 34, 173e181. Shukla, E., Singh, S.S., Singh, P., Mishra, A.K., 2012. Chemotaxonomy of heterocystous cyanobacteria using FAME profiling as species markers. Protoplasma 249, 651e661. Singh, V., 2001. Monograph on Indian Leucas R.Br.. (Dronapushpi) Lamiaceae. Scientific Publishers, Jodhpur. Sinha, S., Ashfaque, A.A., Osman, S.M., 1978. Leucas cephalotes: a new seed oil rich in laballenic acid. Chem. Ind. Lond. 2, 67. Suganya, G., Karthi, S., Shivakumar, M.S., 2014. Larvicidal potential of silver nanoparticles synthesized from Leucas aspera leaf extracts against dengue vector Aedes aegypti. Parasitol. Res. 113, 1673e1679. Sunojkumar, P., Mathew, P., 2009. South Indian Leucas R.Br.. (Lamiaceae): a Taxonomic Monograph. Upchurch, R.G., 2008. Fatty acid unsaturation, mobilization, and regulation in the response of plants to stress. Biotechnol. Lett. 30, 967e977. Walley, J.W., Kliebenstein, D.J., Bostock, R.M., Dehesh, K., 2013. Fatty acids and early detection of pathogens. Curr. Opin. Plant Biol. 16, 520e526. drono, F., Pasquier, E., Deluc, L.G., Marpeau, A.M., Wolff, R.L., Lavialle, O., Pe Aitzetmüller, K., 2001. Fatty acid composition of Pinaceae as taxonomic markers. Lipids 36, 439e451. Wu, J., Seliskar, D.M., Gallagher, J.L., 1998. Stress tolerance in the marsh plant Spartina patens: impact of NaCl on growth and root plasma membrane lipid composition. Physiol. Plant 102, 307e317. Yaeno, T., Matsuda, O., Iba, K., 2004. Role of chloroplast trienoic fatty acids in plant disease defense responses. Plant J. 40, 931e941. Youdim, K.A., Martin, A., Joseph, J.A., 2000. Essential fatty acids and the brain: possible health Implications. Int. J. Dev. Neurosci. 18, 383e399. Zhang, J., Liu, H., Sun, J., Li, B., Zhu, Q., Chen, S., Zhang, H., 2012. Arabidopsis fatty acid desaturase FAD2 is required for salt tolerance during seed germination and early seedling growth. PLoS One 7 (1), e30355.