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Selection of superior Ocimum sanctum L. accessions for industrial application
Industrial Crops & Products 108 (2017) 700–707
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Selection of superior Ocimum sanctum L. accessions for industrial application ⁎
MARK
Parmeshwar Lal Saran , Vandana Tripathy, Ajoy Saha, Kuldeepsingh A. Kalariya, Manish Kumar Suthar, Jitendra Kumar ICAR-Directorate of Medicinal and Aromatic Plants Research, Boriavi 387310, Anand, Gujarat, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Ocimum sanctum Eugenol Chemical characterization Leaf recovery Harvesting stage
Holy basil (Ocimum sanctum L.), a sacred medicinal plant in India is widely used in Indian and other international traditional medicinal systems. Eleven accessions of the plant have been characterized for 21 quantitative and qualitative traits and six essential oil components. Three different harvesting stages of accessions grown in western India were collected for selecting superior accessions with desirable morphological and chemical characters affecting yield and contributing traits for industrial use. The essential oil was extracted by hydro distillation and the characterized by gas chromatograph-mass spectrometry. The colour of the leaves, new branches and inflorescence varied from green to purple-green. The basil accession, DOS-1, resulted in maximum dry leaf recovery (230 g kg−1), total chlorophyll content (1.25 mg g−1), carotenoid content (8.5 mg mL−1) and number of peltate glands. The oil content in green herbage was maximum in DOS-1 (50 g kg−1), followed by DOS-3 (44 g kg−1) at crop maturity. Maximum oil and eugenol yield was also observed in DOS-1 (73 kg ha−1 and 67 kg ha−1). Overall, DOS-1 accession was found superior for leaf recovery, oil yield and eugenol content and therefore, can be used further in crop improvement and commercial cultivation as a new selection.
1. Introduction The basils are largely distributed in Asia, Australia, West Africa and also in some Arabian countries mainly in drier sandy areas (Pistrick, 2001). It is a group of 50–150 species comprising of herbs and shrubs from the tropical regions. The holy basil (Ocimmum sanctum L. syn. Ocimum tenuiflorum) (Family Lamiaceae), is the most sacred herb. It is a native of India and is cultivated largely as a house hold species in India. It is a diploid species having chromosome no. 2n = 32. Leaf types in basils are simple, ovate, elliptic-oblong, obtuse or acute and the leaf margins are usually slightly toothed with entire or sub-serrate or dentate. Both the adaxial and abaxial surfaces are pubescent and dotted with minute glands and slender hairy petioles (Malav et al., 2015). Basil leaves find extensive use in Ayurvedic system of medicine for various ailments including the lowering of plasma glucose (Mukherjee et al., 2006). Essential oils and herbal extracts have attracted a great deal of scientific research interest due to potential as natural flavours. The most common uses of the holy basil include preparation of herbal tea, healing remedies, cosmetics and as preservative (Anbarasu and Vijayalakshmi, 2007). Essential oils of holy basil is valued due to active constituent eugenol that contributes to their therapeutic potential (Kothari et al., 2004). Other constituents in the essential oil include thymol, citrol, geraniol, camphor, linalool, and methyl cinnamate
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Corresponding author. E-mail addresses: [email protected], [email protected] (P.L. Saran).
http://dx.doi.org/10.1016/j.indcrop.2017.07.028 Received 7 April 2017; Received in revised form 13 July 2017; Accepted 14 July 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
(Padalia and Verma, 2011; Singh et al., 2011; Verma et al., 2011). Methyl eugenol (ME), which is chemically known as 4-allylveratrole; 4allyl-1,2- dimethoxybenzene; 1,2- dimethoxy-4-(2-propenyl) benzene; 3,4- dimethoxy-allylbenzene; 3-(3,4- dimethoxyphenyl) prop-L-ene, is the chief aromatic compound in holy basils. The biosynthesis of ME is routed through formation of an essential amino acid, the phenylalanine through caffeic acid and ferulic acid (Herrmann and Weaver 1999). Synthetic ME is now in wide use in perfumery, aromatherapy as well as in processed food industry as flavoring agent (Tan and Nishida, 2012). Recently, antifungal activity of holy basils was documented (Sethi et al., 2013). Eugenol is usually extracted from clove buds (70–85%) as well as leaves and barks of Cinnamomum (20–50%). Although, these plants are rich in eugenol but their cost of commercial extraction is very high (Shasany, 2016). Morphological and chemotypic characterization is the main criterion for selection of suitable Ocimum sanctum variety for herbal industry. The variants with different combinations of purple or green calyces and purple or white corolla are available in India and adjoining regions. These include light green-leaved (Rama tulsi) and purple/dark green-leaved (Shyama tulsi) (Kothari et al., 2004). Oil yield, leaf stage and no. of peltate gland (PG) also play important role in the content of essential oil obtained from plants. Although various workers have reported the variation in the
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2.4. Leaf colour intensity
chemical composition and essential oil content in the Ocimum sp., very few comprehensive studies have been conducted regarding the morphological and chemical characterization of the different accessions to select a superior accession, which can be commercially explored for industrial application. With this background in mind, the current study was undertaken to determine the important morphological parameters to select superior O. sanctum accessions from western regions of India that have better herbage, oil yield and chemical composition of leaf essential oils and the best stage of harvesting.
The intensity of the colour of the leaf, inflorescence and branches was determined based on a scale developed by a panel of five scientists for confirming the observations taken from experimental fields. The intensity of purple or green colour at the maturity stage was measured by the scientists. The aim was to morphologically characterize the accession for the extent to which colour was perceived as crucial factor. The scoring procedure was consummate with the help of check list of odd numbers from 0 to 13 put during the proper stage for intensity of purple colour of new branch, an inflorescence and the leaf including veins, margin and mid rib. For each trait, the score of individual scientists was added and each rank was confirmed into scores on majority basis. The maximum score for all the values were arranged in descending order (Table 2).
2. Materials and methods 2.1. Experimental site The study was conducted at the experimental fields of the Directorate of Medicinal and Aromatic Plants Research (DMAPR), Boriavi, Anand, Gujarat (India) during two consecutive harvesting seasons in the years 2015 and 2016. The experimental farm is located at 22°35′N and 72°55′E at an altitude of about 45.1 m above mean sea level.
2.5. Relationship between peltate gland and age of leaf Leaves were collected from experimental field at three different stages viz., young (fully expanded), recently mature (dark colored) and old (before yellowing). The leaves at tip of the axis to 3rd; 4th–6th and 7th–9th internode, respectively, were used for measuring leaf area using LAM (LAI 3000) and counting no. of PG in 0.5 mm2 area under light microscope at 10 X visualization.
2.2. Plant materials Plants of ten accessions of holy basil collected from different parts of India, were maintained and compared with check variety “Angana”, for inclusion in the present study. The collection sites of accessions and their origin are listed in Table 1. Seeds were sown in the field at DMAPR, Boriavi, Anand (Gujarat) with a spacing of 45 cm × 45 cm. The crop was raised following the standard agronomic practices. Harvesting was done during September–October months of 2015 and 2016. Leaves were collected for analysis at full flowering stage from each accession. Field screening of different accessions was carried out for leaf yield and related traits. The plants selected for analysis were of uniform in age. In the field, three blocks of each germplasm were marked and ten plants in each block were randomly chosen for observations represented as a replication. The values of different observations obtained from these plants were averaged to get the mean value.
2.6. Analysis of the essential oil composition The chemical characterization of the essential oils was performed on a Thermo Focus GC coupled with Thermo Polaris Q single quadruple mass spectrophotometer (MS) detector in Electron Ionization mode and Thermo triplus autosampler on a DB-5MS capillary column (30 m, 0.25 mm id, 0.25 μ film thickness) with the following operating conditions initial oven temperature 60 °C, held for 5 min, then a 5 °C/min tamp to 250 °C and held for 3 min; carrier gas he constant flow @ 1.0 mL min−1, injection volume 0.5 μL (split flow-1:20), the temperature for Inlet, ion source and MS transfer line was 240 °C, 220 °C and 240 °C, respectively. The GC column was coupled directly to the spectrometer in EI mode at 70 eV with the mass range of 40–500 atomic mass unit at 1 scan/s. Individual compounds were identified by mass spectrometry and their identities were confirmed by comparing mass spectra with Mass Spectral Library and literature (Adams, 2007; NIST, 2005).
2.3. Extraction of essential oils The fresh biomass of the tagged plants was harvested at maturity stage for extraction of essential oil. Freshly harvested leaves (1000 g each) were hydro-distilled for 3.0 h in a Clevenger-type apparatus in triplicate. The essential oil layer was separated. The distillate was extracted with diethyl ether and etheral layer was dried over anhydrous sodium sulphate. Ether was distilled off on gently heated water bath and oils were stored in amber colour vials at 4–8 °C for further analysis.
2.7. Economics On an average, a yield of 1843 kg ha−1 dry leaf and 73 kg ha−1 oil was obtained from harvests. The farmer sold its dry leaf at US$ 2.32 kg−1 and oil at US$ 38.88 kg−1 in local market. The material was turned over on alternate days during drying under shed condition and oil was extracted from fresh herbage using Hydro-distillation. The primary data were collected through personal interview using a pre-tested questionnaire. To examine the economics, simple cost accounting method was followed and the financial feasibility was worked out by comparing costs and returns. The prices used in the analysis were averages for the period 2015–16.
Table 1 Geographic information of collection site of different accession. Accession
TC-1 DOS-1 DOS-3 DOS-4 DOS-5 DOS-7 CHES G-1 DEDIYA G-1 DEDIYA S.P. DEDIYA P-1 Angana
Type
Local Selection Local Selection Local Selection Local Selection Local Selection Local Selection Local Selection Local Selection Local Selection Local Selection Variety
State
Gujarat Gujarat Gujarat Gujarat Gujarat Gujarat Gujarat Gujarat Gujarat Gujarat U.P.
Local area
Boriavi Mogar, Anand Boriavi Boriavi Boriavi Boriavi Vejalpur Dediapada Dediapada Dediapada CIMAP Lucknow
Location Lat 0N
Long 0E
22° 22° 22° 22° 22° 22° 22° 21° 21° 21° 26°
72° 73° 72° 72° 72° 72° 73° 73° 73° 73° 80°
61′ 54′ 61′ 61′ 61′ 61′ 41′ 38′ 38′ 38′ 87′
2.8. Statistical analysis
93′ 02′ 93′ 93′ 93′ 93′ 34′ 35′ 35′ 35′ 98′
The statistical analysis of the data was performed using standard statistical procedures. The analysis of variance was done in randomized block design for observations recorded during experiment by using statistical software SAS 9.2 (SAS, 2008). DMRT comparisons among the essential oil, compounds obtained from the accessions including check. The results were presented at 5% level of significance (P = 0.05). The critical difference (CD) values were calculated to compare the various treatment means. 701
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Table 2 Purple and green leaf colour intensity as a morphological trait at plant maturity. Accession
Score
Green and purple colour intensity
Other traits
Dedia P-1
13
Leaf (upper), petiole, new branches and inflorescence are dark purple in colour
Angna DOS-4
11 9
DOS-7
7
Dedia SP-1
5
CHES G-1
3
TC-1, DOS-3 and DOS5 DOS-1 and Dedia G-1
1
Leaf, petiole, new branches and inflorescence are purple in colour Leaves are light purple in colour with medium purple lamina. Petiole and new branches and inflorescence are dark purple in colour Leaves are light purple in colour. Petiole and new branches are medium purple in colour and inflorescence are dark purple in colour. Leaves are green in colour with medium purple veins. Petiole, new branches and inflorescence are medium purple in colour. Leaves are green with slight purple upper side vein only. Petiole, new branches and inflorescence are medium purple in purple. Leaves are green in colour with light purple petiole, new branches and inflorescence colour Leaf, petiole, new branches and inflorescence are green in colour at the maturity
Downward folding of leaves and minimum leaf petiole length – –
0
3. Results and discussion
– – Maximum stem girth and leaf petiole thickness Minimum leaf petiole thickness in DOS-3 and maximum petiole length in TC-1 Minimum stem girth in DOS-1
observed in Angana (1950) which was at par with Dediya P-1 (1886). The highest stem girth was observed in CHES G-1 (18 mm) followed by Dediya P-1 (17 mm). Leaf petiole length was maximum in TC-1 (3.0 mm) whereas the leaf petiole thickness was maximum in CHES G-1 (1.1 mm). The highest biological yields were observed in Dediya P-1 (19,900 kg ha−1), CHES G-1 (19,700 kg ha−1) and Angana (19,700 kg ha−1). DOS-4 (14,400 kg ha−1), on the other hand, showed minimum biological yield. The maximum and minimum green herbage yield were observed in Angana (16,900 kg ha−1) and DOS-4 (10,100 kg ha−1), respectively. Variation for herbage yield and contributing traits in sweet basil germplasm has been recently reported (Saran et al., 2017). However, minimum variance was recorded earlier for fresh herbage yield (Szabo et al., 1996). The highest essential oil and oil yield in green herbage was observed in DOS-1 (50 g kg−1 and 73 kg ha−1), whereas DOS-7 (22 g kg−1, 31 kg ha−1) and DOS-4 (29 g kg−1, 29 kg ha−1) had the lowest oil content. The maximum eugenol elements yield was in DOS-1 (67 kg ha−1) which was at par with DOS-3 (50 kg ha−1) and lowest in DOS-5 (16 kg ha−1). Essential oil content in holy basil var. Krishna and shri tulsi under Pantnagar condition ranged from 18 to 37 g kg−1, respectively (Sethi et al., 2013), which was comparatively less than DOS1 (50 g kg−1). The fresh leaf yield was highest in Angana (8000 kg ha−1) and TC-1 (7900 kg ha−1). The dry leaf yield was highest in DOS-7
3.1. Morphological variation Different holy basil accessions were observed for variation of colours (purple to green) new branch, leaf and inflorescence (Table 2, Fig. 1). Accessions Dediya P-1, Angna, DOS-4, DOS-7 possessed dark purple colour of branch and inflorescence; Dediya SP-1 and CHES G-1 possessed medium purple colour while TC-1, DOS-3 and DOS-5 possessed light purple colour. DOS-1 and Dediya G-1, on the other hand, possessed green colour of leaf and petiole. Upper side of leaf was dark purple in Dediya P-1 whereas, both side of leaf were purple colour in Angna. DOS-4 and DOS-7 possessed light purple colour of leaves with medium purple lamina and petiole whereas, Dediya SP-1, CHES G-1, TC-1, DOS-3 and DOS-5 possessed green colour of leaves with medium purple veins. The high degree of variation among the studied accessions indicating rich diversity was akin to earlier study on the populations from different geographical regions (Malav et al., 2015). The holy basil accessions were evaluated for morphological variations of yield contributing traits at harvesting stage (Table 3). Harvesting was done by cutting the part just above the lignified stem fragment. The plant height in the germplasm ranged from 119 cm in Angna to 75 cm in DOS-4. Plant spread was maximum in Angana (71 cm), and minimum in DOS-1 (52 cm). The maximum no. of branches/plant was in DOS-3 (19) and the highest no. of leaves/plant was
Fig. 1. Morphological variation in different holly basil accession.
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Table 3 Morphological variation for yield contributing traits in eleven accessions of holy basil. Accession
TC-1 DOS-1 DOS-3 DOS-4 DOS-5 DOS-7 CHES G-1 Dediya G-1 Dediya SP-1 Dediya P-1 Angana
Plant height
Plant spread
cm
cm
82gh 85fg 78hi 75i 96cd 96cd 99c 88ef 92de 114b 119a
62bc 52de 57cde 56cde 56cde 58cd 59c 50e 62bc 67ab 71a
No. of branches/ plant
13d 15cd 19a 16c 16bc 12e 16bc 15c 16bc 17b 16bc
No. of leaf/plant
1729de 1865c 1778cd 1874bc 1751d 1413ef 1583e 1167g 1345f 1886b 1950a
Stem girth
Leaf petiole length
Leaf petiole thickness
Biological yield
Green herbage yield
Oil
Oil yield
Eugenol elements Yield
mm
mm
mm
kg ha−1
kg ha−1
g kg−1
kg ha−1
kg ha−1
16bc 13f 15d 15bcd 15cd 15bcd 18a 14de 16b 17a 16bc
3.0a 2.5ab 2.3abc 2.1bc 2.4ab 2.6a 1.9bcd 1.8d 2.1abc 1.9bcd 2.2abc
0.8cd 0.9bc 0.8e 0.8e 0.9b 0.9c 1.1a 0.9bc 1.0a 0.8de 0.9ab
18,500c 18,600c 19,400c 14,400d 17,700cd 18,800c 19,700ab 18,600c 17,100cd 19,900a 19,700ab
13,300cd 14,600bc 14,500bc 10,100e 12,400cd 13,900cd 14,000bcd 12,900cd 11,700de 16,300ab 16,900a
30bcd 50ab 44ab 29cd 17e 22de 27cd 34bcd 34bcd 40abc 34bcd
40c 73a 64ab 29cd 21e 31d 38c 44c 40c 52b 57b
29ef 67a 50b 26f 16g 27f 30def 35de 31def 41cd 44bc
Means with the same letter (superscript) in the columns do not showing significantly different (P = 0.05) − (Duncan Multiple Range Test). Table 4 Variation for leaf yield and contributing traits in eleven accessions of holy basil. Accession
Fresh leaf yield kg ha−1
Dry leaf yield kg ha−1
Leaf recovery g kg−1
Leaf area cm2
Chlorophyll A mg g−1
Chlorophyll B mg g−1
Total Chlorophyll mg g−1
Carotenoids mg mL−1
TC-1 DOS-1 DOS-3 DOS-4 DOS-5 DOS-7 CHES G-1 Dediya G-1 Dediya SP-1 Dediya P-1 Angana
7900a 7900ab 7700bc 5500e 6800cde 5800de 7200bc 7000cd 7000cd 7200bc 8000a
1800bc 1800b 1500cd 1200e 1500cde 2300a 1600c 1300de 1500cde 1600cd 1600cd
230a 230a 200bc 210bc 220ab 230a 230a 180c 210bc 220ab 210bc
9.7bc 7.0e 8.7cd 7.6de 12.6a 7.4e 9.9bc 7.3e 11.7ab 10.2b 9.5bc
0.92b 1.00a 0.84bc 0.78c 0.71cd 0.88bc 0.75cd 0.96ab 0.92b 0.71cd 0.95ab
0.21b 0.24ab 0.19c 0.17cd 0.16d 0.21b 0.16d 0.22b 0.21b 0.17cd 0.26a
1.13bc 1.25a 1.03cd 0.95d 0.87de 1.09bc 0.91d 1.17b 1.13bc 0.88de 1.21ab
7.0cd 8.5a 6.4e 5.4f 4.8g 6.9de 6.2e 8.4ab 7.7bc 5.5f 7.3bc
Means with the same letter (superscript) in the columns do not showing significantly different (P = 0.05) − (Duncan Multiple Range Test).
(2300 kg ha−1) followed by DOS-1 (1800 kg ha−1). The highest leaf recovery was observed in DOS-1 (230 g kg−1), whereas, lowest leaf recovery was observed in Dediya G-1 (180 g kg−1), respectively. The highest leaf area was observed in DOS-5 (12.6 cm2), while minimum in DOS-1 (7.0 cm2). Leaves gave highest chlorophyll and carotenoids content in DOS-1, and lowest in DOS-5 (Table 4). Better leaf yield and recovery in DOS-1 accession indicated better suitability of this accession to green herb and herbal tea industry. Higher dry leaf recovery may be due to lower content of water in leaf or high amount of secondary metabolites. The number of peltate gland (PG) per leaf at different leaf maturity stage in holly basil accessions are presented in Figs. 2 and 3. The maximum leaf area in young leaf stage was observed in Dediya P-1 (6 cm2) followed by DOS-1 (5 cm2). The maximum PG in young leaf stage was observed in DOS-1 (8478) followed by Dediya P-1 (6059), whereas minimum leaf area per leaf was observed in DOS-3 (3329). Similarly, maximum no. of PG per leaf was observed in DOS-1 (11,138 and 12,978) while minimum in DOS-3 and Angna (3360 and 2090) in recently mature and old leaf, respectively. Overall results show that DOS-1 leaves had maximum no. of PG at all the three stages (Fig. 2). Variability for number and types of trichomes (capitate, non-glandular, peltate and different versions and combinations of these) was reported in Lamiaceae genera (Turner et al., 2000) and recognized as the defense-related structures on the aerial epidermis of leaves, stems and floral organs (Wagner, 1991). The internal gland originates from elementary meristem and is associated with the biosynthesis of oils present inside the leaves and stems (Guo et al., 2013). Essential oil glandular trichomes amassed significant quantities of valuable essential oil terpenoids (Shruti et al., 2013). Young basil leaves have been shown to have a higher density of oil glands than older leaves (Sims et al., 2014).
The changes in the essential oil with leaf maturation is due to density of oil glands in sweet basil (Gang et al., 2001). Over all basils are highly cross-pollinated species, therefore, chances of inter and intra-specific hybridization among basil species resulted in morphotypic variation (Tilwari et al., 2013).
3.2. Composition of essential oils The essential oils of different accessions of the O. sanctum were subjected to detailed chemical characterization in order to determine their impact on composition of volatile constituents. Several compounds were identified at onset of maturity (Table 5). Maximum eugenol elements were recorded in DOS-1 (970 g kg−1) which was at par with DOS-4 (940 g kg−1). In earlier investigations, methyl eugenol was reported as the major constituent of holy basil herb, leaf, stem and inflorescence (725 g kg−1, 753 g kg−1, 837 g kg−1 and 652 g kg−1) in respective concentration (55 g kg−1, 64 g kg−1, 27 g kg−1 and 12 g kg−1) in each oil (Kothari et al., 2005). The morphologically distinct ‘Rama’ and ‘Shyama’ types from India and many chemotypes within them have been reported to contain eugenol and methyl eugenol (Archana et al., 2013). As compared to the highest average eugenol share (913 g kg−1) in DOS-1 accession in our study, the earlier reported highest share of eugenol in clove basil under Uttarakhand conditions was only 775 g kg−1 (Sethi et al., 2013). The maximum Azuline was found in Dadiya P-1 (110 g kg−1) followed by Dadiya SP-1 (95 g kg−1) but was absent in DOS-1. Similarly, maximum share of β-Caryophyllene was observed in CHES G-1 and Dedia G-1 (182 g kg−1), but nil in DOS-1 and Angana. β-Caryophyllene was the second most dominant constituent from whole herb, leaf, stem and inflorescence in respective concentration (55 g kg−1, 64 g kg−1, 27 g kg−1 and 120 g kg−1) in 703
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Fig. 2. Relationship between no. of peltate gland (PG) and age of leaf.
each oil (Kothari et al., 2005). Out of the 11 accessions, Alfa-cubebene and delta-3-carene were absent in five accessions, namely, DOS-1, DOS3, CHES G-1, Dedia G-1 and Dadiya SP-1. In the remaining six accessions, their corresponding concentration was highest in Angana (124 g kg−1 and 27 g kg−1) followed by DOS-7 (35 g kg−1 and 5 g kg−1). Eugenol was the most dominant constituent followed by βCaryophyllene in the tested accessions. With the advancement of harvesting stages, the maximum share of eugenol elements at the onset of flowering, full flowering and crop maturity was 860 g kg−1, 890 g kg−1 and 990 g kg−1 in DOS-1 (increasing trend) while minimum share was observed in Angana (160 g kg−1, 580 g kg−1 and 770 g kg−1). However, reverse trend was observed for β-Caryophyllene in DOS-1 (100 g kg−1, 90 g kg−1 and 00 g kg−1) and Angana (200 g kg−1, 100 g kg−1 and 00 g kg−1), respectively (Figs. 4 and 5). The major oil constituents of O. tenuiflorum grown in Australia (methyl chavicol 870 g kg−1), Bangladesh (eugenol 417 g kg−1 and sesquiterpene components 459 g kg−1 in green type and eugenol 775 g kg−1 in purple type), Cuba (eugenol 343 g kg−1, β-elemene 180 g kg−1 and β-caryophyllene 231 g kg−1), Germany (eugenol 242 g kg−1, α-bisabolene 106 g kg−1, β-bisabolene 154 g kg−1 and methyl chavicol 116 g kg−1) and India (eugenol 350–530 g kg−1) are distinctly different from the chemotype reported in this study (Laakso et al., 1990; Pino et al., 1998; Raju et al., 1999). In earlier study, the level of eugenol and methyl eugenol in holy basil declined with progressive maturation of the plant leaves (Dey and Choudhuri, 1983). Eugenol, β-caryophyllene, E-methyl cinnamate and (trans)-β-guaiene were the most abundant components identified from three accessions (Sims et al., 2014). Influence of planting date and harvesting was recorded in three O. tenuiflorum accessions in Alabama. In accession PI 652056, the level of eugenol increased with a delay in harvest time, while, accession PI 652057, the level of β-caryophyllene was high at the 30-day harvest, but decreased significantly by the time of the 60-day harvest when eugenol became the dominant essential oil constituent (Sims et al., 2014). 3.3. Economics
Fig. 3. The no. of Peltate Glands (PG) per leaf at different leaf maturity stage.
Clove, is a major source of eugenol in India but is not produced in 704
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Table 5 Chemotypic composition of essential oils (g kg−1) at crop maturity stage in eleven accessions of holy basil. Accession
Eugenol element
Azuline
β-Caryophyllene
α-Cubebene
Delta 3 carene
α-humulene
TC-1 DOS-1 DOS-3 DOS-4 DOS-5 DOS-7 CHES G-1 Dediya G-1 Dediya SP-1 Dediya P-1 Angana
730j 990a 780g 940b 730j 880c 800e 800d 780h 790f 770i
39de 0.0 87bc 38de 89bc 64cd 3ef 4ef 95b 110a 5.0ef
119d 0.0 117e 0.0 139c 5.0h 182a 182b 116f 75g 0.0
13d 0.0 0.0 3.0e 22c 35b 0.0 0.0 0.0 2.0ef 124a
3.0c 0.0 0.0 4.0c 2.0d 5.0b 0.0 0.0 0.0 1.0d 27a
0.0 5.0c 6.0bc 0.0 2.0f 4.0d 8.0ab 9.0a 5.0c 2.0f 3.0e
Means with the same letter (superscript) in the columns do not showing significantly different (P = 0.05) − (Duncan Multiple Range Test). Fig. 4. Relationship between eugenol element and βCaryophyllene at different leaf harvesting stage.
Fig. 5. Chromatogram of volatile compound identified in accession (A) DOS-1 at onset of flowering; (B) DOS-1 at full flowering; (C) DOS-1 at maturity; (D) Angana at onset of flowering; (E) Angana at full flowering and (F) Angana at maturity from GC/MS.
necessitates the need to find out some cheaper alternatives like Ocimum species. In this study, basil accession DOS-1 has been found to be rich in eugenol content. On an average, a farmer can get gross returns from dry leaf and essential oil from fresh herb marketing approximately US$
sufficient quantity to meet the requirement of the country. Eugenol is usually extracted from the clove buds (70–85%) as well as from the leaves and barks of Cinnamomum (20–50%) (Shasany, 2016). Further, the high cost involved in the extraction of eugenol from clove, 705
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Fig. 6. Economics of Accession DOS-1 for leaf and oil yield/hectare.
Fig. 7. Field view of DOS-1 at farmer’s field as a Front-Line Demonstration.
4281 ha−1 and US$ 2838 ha−1 and B:C ratio over total cost was 1.6 and 2.2, respectively (Fig. 6). Compared with clove, Ocimum species can be grown in varied soil and climatic conditions. The crop is ready for harvesting in 90–95 days after planting and subsequent harvests can be taken in 60–65 days interval (Fig. 7). Besides, cost of production of different Ocimum species is far less compared with clove (Smitha and Tripathy, 2016). The holy basil is cheaper source for commercial extraction of eugenol (Mukherji, 1987). It is thus concluded that the holy basil accession DOS-1 can be commercially cultivated as potential sources of eugenol with good returns.
herbal medicines and other use applications. Competing interests The authors declare that they have no competing interest. Authors’ contribution Dr. Parmeshwar Lal Saran was involved in conception, design of the experiments and acquisition of data. He has collected all data and extracted the essential oil from the plants. Dr. Vandana Tripathy analyzed the essential oil by GCMS for the first two stages and Dr. Ajoy Saha for last maturity stage. Dr. Kalariya has analyzed chlorophyll and carotenoids content and Dr. Manish Kumar Suthar counted PG in all the accessions. Dr. Jitendra Kumar, Director, provided all the facilities and guidance.
4. Conclusion Different harvesting stages of O. sanctum accessions grown in western India have been investigated for desirable morphological and chemotypic variations. Maximum oil per cent in green herbage, dry leaf recovery, total chlorophyll, essential oil, carotenoids and eugenol elements was observed in DOS-1 accession at crop maturity. Similarly, the DOS-1 leaves contained maximum no. of peltate glands (PG) at all three stages. The maximum share of eugenol/methyl eugenol was observed in DOS-1 at onset of flowering, full flowering and crop maturity in increasing order with advancement of harvesting stages, while reverse trend was observed for β-Caryophyllene. Overall, holy basil and in particular DOS-1 accession was found superior and thus, can be considered for further improvement. Instead of costly clove, O. sanctum (basil) leaves offer cost effective substitute for herbal tea industry,
References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry. Allured Publishing Corporation, Carol Stream, Illinois. Anbarasu, K., Vijayalakshmi, G., 2007. Improved shelf life of protein-rich tofu using Ocimum sanctum (tulsi) extracts to benefit Indian rural population. J. Food Sci. 72, 300–305. Archana, P.R., Ashok, K., Dutta, M., 2013. Chemical characterization of aroma compounds in essential oil isolated from Holy Basil (Ocimum tenuiflorum L.) grown in India. Genet. Resour. Crop Evol. 60, 1727–1735. Dey, B.B., Choudhuri, M.A., 1983. Effect of leaf development stage on changes in essential oil of Ocimum sanctum L. Biochem. Physiol. Pflazen 178, 331–335.
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constituents of the leaves of Ocimum sanctum L. J. Essent. Oil Res. 11, 159–161. SAS, 2008. SAS Institute Inc. 2008. SAS/STAT® 9.2 User’s Guide. SAS Institute Inc, Cary, NC. https://support.sas.com/documentation/cdl/en/statugstatmodel/61751/PDF/ default/statugstatmodel.pdf. Saran, P.L., Tripathy, V., Meena, R.P., Kumar, J., Vasara, R.P., 2017. Chemotypic characterization and development of morphological markers in Ocimum basilicum L. germplasm. Sci. Hortic. 215, 164–171. Sethi, S., Praksha Om Chandra, M., Punetha, H., Pant, A.K., 2013. Antifungal activity of essential oils of some ocimum species collected from different locations of Uttarakhand. Indian J. Nat. Prod. Res. 4 (4), 392–397. Shasany, A.K., 2016. The holy basil (Ocimum sanctum L.) and its genome. Indian J. Hist. Sci. 51 (2), 343–350. Sims, C.A., Juliani, H.R., Mentreddy, S.R., Simon, J.E., 2014. Essential oils in holy basil (Ocimum tenuiflorum L.) as influenced by planting dates and harvest times in North Alabama. J. Med. Active Plants 2 (3), 33–41. Singh, S.K., Anand, A., Verma, S.K., Siddiqui, M.A., Mathur, A., Saklani, S., 2011. Analysis of phytochemical and antioxidant potential of Ocimum kilimandscharicum Linn. Int. Curr. Pharma Res. J. 3, 40–46. Smitha, G.R., Tripathy, V., 2016. Seasonal variation in the essential oils extracted from leaves and inflorescence of different Ocimum species grown in Western plains of India. Ind. Crops Prod. 94, 52–64. Szabo, K., Nemeth, E., Prapzha, L., Bernath, J., Pank, F., 1996. Morphological and chemical variability of basil genatypes. International Symposium on Breeding Research on Medicinal and Aromatic Plants Quedlinbury Germany vol. 2 (1), pp. 76–79. Tan, K.H., Nishida, R., 2012. Methyl eugenol: its occurrence, distribution, and role in nature, especially in relation to insect behavior and pollination. J. Insect Sci. 12, 56 (insectscience.org/12.56). Tilwari, A., Tamrakar, K., Sharma, R., 2013. Use of random amplified polymorphic DNA (RAPD) for assessing genetic diversity of Ocimum sanctum (Krishna tulsi) from different environments of Central India. J. Med. Plants Res. 7 (24), 1800–1808. Turner, G.W., Gershenzon, J., Croteau, R.B., 2000. Development of peltate glandular trichomes of peppermint. Plant Physol. 124, 665–679. Wagner, G.J., 1991. Secreting glandular trichomes: more than just hairs. Plant Physiol. 96, 675–679.
Gang, D.R., Wang, J., Dudareva, N., Nam, K.H., Simon, J.E., Lewinsohn, E., Pichersky, E., 2001. An investigation of the storage and biosynthesis of phenylpropenes in sweet basil. Plant Physiol. 125 (2), 539–555. Guo, J., Yuan, Y., Liu, Z., Zhu, J., 2013. Development and structure of internal glands and external glandular trichomes in Pogostemon cablin. PLoS One 8 (10), e77862. http:// dx.doi.org/10.1371/journal.pone.0077862. Herrmann, K.M., Weaver, L.M., 1999. The shikimate pathway. Ann. Rev. Plant Physiol. Plant Mol. Biol. 50, 473–503. Kothari, S.K., Bhattacharya, A.K., Ramesh, S., 2004. Essential oil yield and quality of methyl eugenol rich Ocimum tenuiflorum L.f. (syn Ocimum sanctum L.) grown in south India as influenced by method of harvest. J. Chromatogr. A 1054, 67–72. Kothari, S.K., Bhattacharya, A.K., Ramesh, S., Garg, S.N., Khanuja, S.P.S., 2005. Volatile constituents in oil from different plant parts of methyl eugenol-rich Ocimum tenuiflorum L.f. syn. O. sanctum L.) grown in South India. J. Essent. Oil Res. 17 (6), 656–658. Laakso, L., Seppanen-Laakso, T., Hermann-Wolf, B., Knobloch, K., 1990. Constituents of the essential oil from the Holy basil or Tulsi basil, Ocimum sanctum. Planta Med. 56, 527. Malav, P., Pandey, A., Bhatt, K.C., Gopala Krishnan, S., Bisht, I.S., 2015. Morphological variability in holy basil (Ocimum tenuiflorum L.) from India. Genet. Resour. Crop. Evol. 62, 1245–1256. Mukherjee, P.K., Maiti, K., Mukherjee, K., Houghton, P.J., 2006. Leads from Indian medicinal plants with hypoglycemic potentials. J. Ethnopharmacol. 106, 1–28. Mukherji, S.P., 1987. Ocimum-a cheap source of Eugenol. Science Reporter, p. 599. 31. Eugenol-rich Ocimum variety released. CSIR NEWS 45. PID CSIR, New Delhi, pp. 256. NIST, 2005. Mass Spectral Library. NIST Mass Spectral Search Program (NIST 05, Version 2.0d). The NIST Mass Spectrometry Data Center, Gaithersburg, MD. Padalia, R.C., Verma, R.S., 2011. Comparative volatile oil composition of four Ocimum species from northern India. Nat. Prod. Lett. 25, 569–575. Pino, J.A., Rosado, A., Rodriguez, M., Garcia, D., 1998. Composition of the essential oil of Ocimum tenuifl orum L. grown in Cuba. J. Essent. Oil Res. 10, 437–438. Pistrick, K., 2001. Ocimum. In: In: Hanelt, P., Institute of Plant Genetics and Crop Plant Research (Eds.), Mansfeld́ s Encyclopedia of Agricultural and Horticultural Crops, vol. 4. Springer, pp. 1954–1960. Raju, P.M., Ali, M., Velasco-Negueruela, A., Perez-Alonso, M.J., 1999. Volatile
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Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
Selection of superior Ocimum sanctum L. accessions for industrial application ⁎
MARK
Parmeshwar Lal Saran , Vandana Tripathy, Ajoy Saha, Kuldeepsingh A. Kalariya, Manish Kumar Suthar, Jitendra Kumar ICAR-Directorate of Medicinal and Aromatic Plants Research, Boriavi 387310, Anand, Gujarat, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Ocimum sanctum Eugenol Chemical characterization Leaf recovery Harvesting stage
Holy basil (Ocimum sanctum L.), a sacred medicinal plant in India is widely used in Indian and other international traditional medicinal systems. Eleven accessions of the plant have been characterized for 21 quantitative and qualitative traits and six essential oil components. Three different harvesting stages of accessions grown in western India were collected for selecting superior accessions with desirable morphological and chemical characters affecting yield and contributing traits for industrial use. The essential oil was extracted by hydro distillation and the characterized by gas chromatograph-mass spectrometry. The colour of the leaves, new branches and inflorescence varied from green to purple-green. The basil accession, DOS-1, resulted in maximum dry leaf recovery (230 g kg−1), total chlorophyll content (1.25 mg g−1), carotenoid content (8.5 mg mL−1) and number of peltate glands. The oil content in green herbage was maximum in DOS-1 (50 g kg−1), followed by DOS-3 (44 g kg−1) at crop maturity. Maximum oil and eugenol yield was also observed in DOS-1 (73 kg ha−1 and 67 kg ha−1). Overall, DOS-1 accession was found superior for leaf recovery, oil yield and eugenol content and therefore, can be used further in crop improvement and commercial cultivation as a new selection.
1. Introduction The basils are largely distributed in Asia, Australia, West Africa and also in some Arabian countries mainly in drier sandy areas (Pistrick, 2001). It is a group of 50–150 species comprising of herbs and shrubs from the tropical regions. The holy basil (Ocimmum sanctum L. syn. Ocimum tenuiflorum) (Family Lamiaceae), is the most sacred herb. It is a native of India and is cultivated largely as a house hold species in India. It is a diploid species having chromosome no. 2n = 32. Leaf types in basils are simple, ovate, elliptic-oblong, obtuse or acute and the leaf margins are usually slightly toothed with entire or sub-serrate or dentate. Both the adaxial and abaxial surfaces are pubescent and dotted with minute glands and slender hairy petioles (Malav et al., 2015). Basil leaves find extensive use in Ayurvedic system of medicine for various ailments including the lowering of plasma glucose (Mukherjee et al., 2006). Essential oils and herbal extracts have attracted a great deal of scientific research interest due to potential as natural flavours. The most common uses of the holy basil include preparation of herbal tea, healing remedies, cosmetics and as preservative (Anbarasu and Vijayalakshmi, 2007). Essential oils of holy basil is valued due to active constituent eugenol that contributes to their therapeutic potential (Kothari et al., 2004). Other constituents in the essential oil include thymol, citrol, geraniol, camphor, linalool, and methyl cinnamate
⁎
Corresponding author. E-mail addresses: [email protected], [email protected] (P.L. Saran).
http://dx.doi.org/10.1016/j.indcrop.2017.07.028 Received 7 April 2017; Received in revised form 13 July 2017; Accepted 14 July 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
(Padalia and Verma, 2011; Singh et al., 2011; Verma et al., 2011). Methyl eugenol (ME), which is chemically known as 4-allylveratrole; 4allyl-1,2- dimethoxybenzene; 1,2- dimethoxy-4-(2-propenyl) benzene; 3,4- dimethoxy-allylbenzene; 3-(3,4- dimethoxyphenyl) prop-L-ene, is the chief aromatic compound in holy basils. The biosynthesis of ME is routed through formation of an essential amino acid, the phenylalanine through caffeic acid and ferulic acid (Herrmann and Weaver 1999). Synthetic ME is now in wide use in perfumery, aromatherapy as well as in processed food industry as flavoring agent (Tan and Nishida, 2012). Recently, antifungal activity of holy basils was documented (Sethi et al., 2013). Eugenol is usually extracted from clove buds (70–85%) as well as leaves and barks of Cinnamomum (20–50%). Although, these plants are rich in eugenol but their cost of commercial extraction is very high (Shasany, 2016). Morphological and chemotypic characterization is the main criterion for selection of suitable Ocimum sanctum variety for herbal industry. The variants with different combinations of purple or green calyces and purple or white corolla are available in India and adjoining regions. These include light green-leaved (Rama tulsi) and purple/dark green-leaved (Shyama tulsi) (Kothari et al., 2004). Oil yield, leaf stage and no. of peltate gland (PG) also play important role in the content of essential oil obtained from plants. Although various workers have reported the variation in the
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2.4. Leaf colour intensity
chemical composition and essential oil content in the Ocimum sp., very few comprehensive studies have been conducted regarding the morphological and chemical characterization of the different accessions to select a superior accession, which can be commercially explored for industrial application. With this background in mind, the current study was undertaken to determine the important morphological parameters to select superior O. sanctum accessions from western regions of India that have better herbage, oil yield and chemical composition of leaf essential oils and the best stage of harvesting.
The intensity of the colour of the leaf, inflorescence and branches was determined based on a scale developed by a panel of five scientists for confirming the observations taken from experimental fields. The intensity of purple or green colour at the maturity stage was measured by the scientists. The aim was to morphologically characterize the accession for the extent to which colour was perceived as crucial factor. The scoring procedure was consummate with the help of check list of odd numbers from 0 to 13 put during the proper stage for intensity of purple colour of new branch, an inflorescence and the leaf including veins, margin and mid rib. For each trait, the score of individual scientists was added and each rank was confirmed into scores on majority basis. The maximum score for all the values were arranged in descending order (Table 2).
2. Materials and methods 2.1. Experimental site The study was conducted at the experimental fields of the Directorate of Medicinal and Aromatic Plants Research (DMAPR), Boriavi, Anand, Gujarat (India) during two consecutive harvesting seasons in the years 2015 and 2016. The experimental farm is located at 22°35′N and 72°55′E at an altitude of about 45.1 m above mean sea level.
2.5. Relationship between peltate gland and age of leaf Leaves were collected from experimental field at three different stages viz., young (fully expanded), recently mature (dark colored) and old (before yellowing). The leaves at tip of the axis to 3rd; 4th–6th and 7th–9th internode, respectively, were used for measuring leaf area using LAM (LAI 3000) and counting no. of PG in 0.5 mm2 area under light microscope at 10 X visualization.
2.2. Plant materials Plants of ten accessions of holy basil collected from different parts of India, were maintained and compared with check variety “Angana”, for inclusion in the present study. The collection sites of accessions and their origin are listed in Table 1. Seeds were sown in the field at DMAPR, Boriavi, Anand (Gujarat) with a spacing of 45 cm × 45 cm. The crop was raised following the standard agronomic practices. Harvesting was done during September–October months of 2015 and 2016. Leaves were collected for analysis at full flowering stage from each accession. Field screening of different accessions was carried out for leaf yield and related traits. The plants selected for analysis were of uniform in age. In the field, three blocks of each germplasm were marked and ten plants in each block were randomly chosen for observations represented as a replication. The values of different observations obtained from these plants were averaged to get the mean value.
2.6. Analysis of the essential oil composition The chemical characterization of the essential oils was performed on a Thermo Focus GC coupled with Thermo Polaris Q single quadruple mass spectrophotometer (MS) detector in Electron Ionization mode and Thermo triplus autosampler on a DB-5MS capillary column (30 m, 0.25 mm id, 0.25 μ film thickness) with the following operating conditions initial oven temperature 60 °C, held for 5 min, then a 5 °C/min tamp to 250 °C and held for 3 min; carrier gas he constant flow @ 1.0 mL min−1, injection volume 0.5 μL (split flow-1:20), the temperature for Inlet, ion source and MS transfer line was 240 °C, 220 °C and 240 °C, respectively. The GC column was coupled directly to the spectrometer in EI mode at 70 eV with the mass range of 40–500 atomic mass unit at 1 scan/s. Individual compounds were identified by mass spectrometry and their identities were confirmed by comparing mass spectra with Mass Spectral Library and literature (Adams, 2007; NIST, 2005).
2.3. Extraction of essential oils The fresh biomass of the tagged plants was harvested at maturity stage for extraction of essential oil. Freshly harvested leaves (1000 g each) were hydro-distilled for 3.0 h in a Clevenger-type apparatus in triplicate. The essential oil layer was separated. The distillate was extracted with diethyl ether and etheral layer was dried over anhydrous sodium sulphate. Ether was distilled off on gently heated water bath and oils were stored in amber colour vials at 4–8 °C for further analysis.
2.7. Economics On an average, a yield of 1843 kg ha−1 dry leaf and 73 kg ha−1 oil was obtained from harvests. The farmer sold its dry leaf at US$ 2.32 kg−1 and oil at US$ 38.88 kg−1 in local market. The material was turned over on alternate days during drying under shed condition and oil was extracted from fresh herbage using Hydro-distillation. The primary data were collected through personal interview using a pre-tested questionnaire. To examine the economics, simple cost accounting method was followed and the financial feasibility was worked out by comparing costs and returns. The prices used in the analysis were averages for the period 2015–16.
Table 1 Geographic information of collection site of different accession. Accession
TC-1 DOS-1 DOS-3 DOS-4 DOS-5 DOS-7 CHES G-1 DEDIYA G-1 DEDIYA S.P. DEDIYA P-1 Angana
Type
Local Selection Local Selection Local Selection Local Selection Local Selection Local Selection Local Selection Local Selection Local Selection Local Selection Variety
State
Gujarat Gujarat Gujarat Gujarat Gujarat Gujarat Gujarat Gujarat Gujarat Gujarat U.P.
Local area
Boriavi Mogar, Anand Boriavi Boriavi Boriavi Boriavi Vejalpur Dediapada Dediapada Dediapada CIMAP Lucknow
Location Lat 0N
Long 0E
22° 22° 22° 22° 22° 22° 22° 21° 21° 21° 26°
72° 73° 72° 72° 72° 72° 73° 73° 73° 73° 80°
61′ 54′ 61′ 61′ 61′ 61′ 41′ 38′ 38′ 38′ 87′
2.8. Statistical analysis
93′ 02′ 93′ 93′ 93′ 93′ 34′ 35′ 35′ 35′ 98′
The statistical analysis of the data was performed using standard statistical procedures. The analysis of variance was done in randomized block design for observations recorded during experiment by using statistical software SAS 9.2 (SAS, 2008). DMRT comparisons among the essential oil, compounds obtained from the accessions including check. The results were presented at 5% level of significance (P = 0.05). The critical difference (CD) values were calculated to compare the various treatment means. 701
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Table 2 Purple and green leaf colour intensity as a morphological trait at plant maturity. Accession
Score
Green and purple colour intensity
Other traits
Dedia P-1
13
Leaf (upper), petiole, new branches and inflorescence are dark purple in colour
Angna DOS-4
11 9
DOS-7
7
Dedia SP-1
5
CHES G-1
3
TC-1, DOS-3 and DOS5 DOS-1 and Dedia G-1
1
Leaf, petiole, new branches and inflorescence are purple in colour Leaves are light purple in colour with medium purple lamina. Petiole and new branches and inflorescence are dark purple in colour Leaves are light purple in colour. Petiole and new branches are medium purple in colour and inflorescence are dark purple in colour. Leaves are green in colour with medium purple veins. Petiole, new branches and inflorescence are medium purple in colour. Leaves are green with slight purple upper side vein only. Petiole, new branches and inflorescence are medium purple in purple. Leaves are green in colour with light purple petiole, new branches and inflorescence colour Leaf, petiole, new branches and inflorescence are green in colour at the maturity
Downward folding of leaves and minimum leaf petiole length – –
0
3. Results and discussion
– – Maximum stem girth and leaf petiole thickness Minimum leaf petiole thickness in DOS-3 and maximum petiole length in TC-1 Minimum stem girth in DOS-1
observed in Angana (1950) which was at par with Dediya P-1 (1886). The highest stem girth was observed in CHES G-1 (18 mm) followed by Dediya P-1 (17 mm). Leaf petiole length was maximum in TC-1 (3.0 mm) whereas the leaf petiole thickness was maximum in CHES G-1 (1.1 mm). The highest biological yields were observed in Dediya P-1 (19,900 kg ha−1), CHES G-1 (19,700 kg ha−1) and Angana (19,700 kg ha−1). DOS-4 (14,400 kg ha−1), on the other hand, showed minimum biological yield. The maximum and minimum green herbage yield were observed in Angana (16,900 kg ha−1) and DOS-4 (10,100 kg ha−1), respectively. Variation for herbage yield and contributing traits in sweet basil germplasm has been recently reported (Saran et al., 2017). However, minimum variance was recorded earlier for fresh herbage yield (Szabo et al., 1996). The highest essential oil and oil yield in green herbage was observed in DOS-1 (50 g kg−1 and 73 kg ha−1), whereas DOS-7 (22 g kg−1, 31 kg ha−1) and DOS-4 (29 g kg−1, 29 kg ha−1) had the lowest oil content. The maximum eugenol elements yield was in DOS-1 (67 kg ha−1) which was at par with DOS-3 (50 kg ha−1) and lowest in DOS-5 (16 kg ha−1). Essential oil content in holy basil var. Krishna and shri tulsi under Pantnagar condition ranged from 18 to 37 g kg−1, respectively (Sethi et al., 2013), which was comparatively less than DOS1 (50 g kg−1). The fresh leaf yield was highest in Angana (8000 kg ha−1) and TC-1 (7900 kg ha−1). The dry leaf yield was highest in DOS-7
3.1. Morphological variation Different holy basil accessions were observed for variation of colours (purple to green) new branch, leaf and inflorescence (Table 2, Fig. 1). Accessions Dediya P-1, Angna, DOS-4, DOS-7 possessed dark purple colour of branch and inflorescence; Dediya SP-1 and CHES G-1 possessed medium purple colour while TC-1, DOS-3 and DOS-5 possessed light purple colour. DOS-1 and Dediya G-1, on the other hand, possessed green colour of leaf and petiole. Upper side of leaf was dark purple in Dediya P-1 whereas, both side of leaf were purple colour in Angna. DOS-4 and DOS-7 possessed light purple colour of leaves with medium purple lamina and petiole whereas, Dediya SP-1, CHES G-1, TC-1, DOS-3 and DOS-5 possessed green colour of leaves with medium purple veins. The high degree of variation among the studied accessions indicating rich diversity was akin to earlier study on the populations from different geographical regions (Malav et al., 2015). The holy basil accessions were evaluated for morphological variations of yield contributing traits at harvesting stage (Table 3). Harvesting was done by cutting the part just above the lignified stem fragment. The plant height in the germplasm ranged from 119 cm in Angna to 75 cm in DOS-4. Plant spread was maximum in Angana (71 cm), and minimum in DOS-1 (52 cm). The maximum no. of branches/plant was in DOS-3 (19) and the highest no. of leaves/plant was
Fig. 1. Morphological variation in different holly basil accession.
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Table 3 Morphological variation for yield contributing traits in eleven accessions of holy basil. Accession
TC-1 DOS-1 DOS-3 DOS-4 DOS-5 DOS-7 CHES G-1 Dediya G-1 Dediya SP-1 Dediya P-1 Angana
Plant height
Plant spread
cm
cm
82gh 85fg 78hi 75i 96cd 96cd 99c 88ef 92de 114b 119a
62bc 52de 57cde 56cde 56cde 58cd 59c 50e 62bc 67ab 71a
No. of branches/ plant
13d 15cd 19a 16c 16bc 12e 16bc 15c 16bc 17b 16bc
No. of leaf/plant
1729de 1865c 1778cd 1874bc 1751d 1413ef 1583e 1167g 1345f 1886b 1950a
Stem girth
Leaf petiole length
Leaf petiole thickness
Biological yield
Green herbage yield
Oil
Oil yield
Eugenol elements Yield
mm
mm
mm
kg ha−1
kg ha−1
g kg−1
kg ha−1
kg ha−1
16bc 13f 15d 15bcd 15cd 15bcd 18a 14de 16b 17a 16bc
3.0a 2.5ab 2.3abc 2.1bc 2.4ab 2.6a 1.9bcd 1.8d 2.1abc 1.9bcd 2.2abc
0.8cd 0.9bc 0.8e 0.8e 0.9b 0.9c 1.1a 0.9bc 1.0a 0.8de 0.9ab
18,500c 18,600c 19,400c 14,400d 17,700cd 18,800c 19,700ab 18,600c 17,100cd 19,900a 19,700ab
13,300cd 14,600bc 14,500bc 10,100e 12,400cd 13,900cd 14,000bcd 12,900cd 11,700de 16,300ab 16,900a
30bcd 50ab 44ab 29cd 17e 22de 27cd 34bcd 34bcd 40abc 34bcd
40c 73a 64ab 29cd 21e 31d 38c 44c 40c 52b 57b
29ef 67a 50b 26f 16g 27f 30def 35de 31def 41cd 44bc
Means with the same letter (superscript) in the columns do not showing significantly different (P = 0.05) − (Duncan Multiple Range Test). Table 4 Variation for leaf yield and contributing traits in eleven accessions of holy basil. Accession
Fresh leaf yield kg ha−1
Dry leaf yield kg ha−1
Leaf recovery g kg−1
Leaf area cm2
Chlorophyll A mg g−1
Chlorophyll B mg g−1
Total Chlorophyll mg g−1
Carotenoids mg mL−1
TC-1 DOS-1 DOS-3 DOS-4 DOS-5 DOS-7 CHES G-1 Dediya G-1 Dediya SP-1 Dediya P-1 Angana
7900a 7900ab 7700bc 5500e 6800cde 5800de 7200bc 7000cd 7000cd 7200bc 8000a
1800bc 1800b 1500cd 1200e 1500cde 2300a 1600c 1300de 1500cde 1600cd 1600cd
230a 230a 200bc 210bc 220ab 230a 230a 180c 210bc 220ab 210bc
9.7bc 7.0e 8.7cd 7.6de 12.6a 7.4e 9.9bc 7.3e 11.7ab 10.2b 9.5bc
0.92b 1.00a 0.84bc 0.78c 0.71cd 0.88bc 0.75cd 0.96ab 0.92b 0.71cd 0.95ab
0.21b 0.24ab 0.19c 0.17cd 0.16d 0.21b 0.16d 0.22b 0.21b 0.17cd 0.26a
1.13bc 1.25a 1.03cd 0.95d 0.87de 1.09bc 0.91d 1.17b 1.13bc 0.88de 1.21ab
7.0cd 8.5a 6.4e 5.4f 4.8g 6.9de 6.2e 8.4ab 7.7bc 5.5f 7.3bc
Means with the same letter (superscript) in the columns do not showing significantly different (P = 0.05) − (Duncan Multiple Range Test).
(2300 kg ha−1) followed by DOS-1 (1800 kg ha−1). The highest leaf recovery was observed in DOS-1 (230 g kg−1), whereas, lowest leaf recovery was observed in Dediya G-1 (180 g kg−1), respectively. The highest leaf area was observed in DOS-5 (12.6 cm2), while minimum in DOS-1 (7.0 cm2). Leaves gave highest chlorophyll and carotenoids content in DOS-1, and lowest in DOS-5 (Table 4). Better leaf yield and recovery in DOS-1 accession indicated better suitability of this accession to green herb and herbal tea industry. Higher dry leaf recovery may be due to lower content of water in leaf or high amount of secondary metabolites. The number of peltate gland (PG) per leaf at different leaf maturity stage in holly basil accessions are presented in Figs. 2 and 3. The maximum leaf area in young leaf stage was observed in Dediya P-1 (6 cm2) followed by DOS-1 (5 cm2). The maximum PG in young leaf stage was observed in DOS-1 (8478) followed by Dediya P-1 (6059), whereas minimum leaf area per leaf was observed in DOS-3 (3329). Similarly, maximum no. of PG per leaf was observed in DOS-1 (11,138 and 12,978) while minimum in DOS-3 and Angna (3360 and 2090) in recently mature and old leaf, respectively. Overall results show that DOS-1 leaves had maximum no. of PG at all the three stages (Fig. 2). Variability for number and types of trichomes (capitate, non-glandular, peltate and different versions and combinations of these) was reported in Lamiaceae genera (Turner et al., 2000) and recognized as the defense-related structures on the aerial epidermis of leaves, stems and floral organs (Wagner, 1991). The internal gland originates from elementary meristem and is associated with the biosynthesis of oils present inside the leaves and stems (Guo et al., 2013). Essential oil glandular trichomes amassed significant quantities of valuable essential oil terpenoids (Shruti et al., 2013). Young basil leaves have been shown to have a higher density of oil glands than older leaves (Sims et al., 2014).
The changes in the essential oil with leaf maturation is due to density of oil glands in sweet basil (Gang et al., 2001). Over all basils are highly cross-pollinated species, therefore, chances of inter and intra-specific hybridization among basil species resulted in morphotypic variation (Tilwari et al., 2013).
3.2. Composition of essential oils The essential oils of different accessions of the O. sanctum were subjected to detailed chemical characterization in order to determine their impact on composition of volatile constituents. Several compounds were identified at onset of maturity (Table 5). Maximum eugenol elements were recorded in DOS-1 (970 g kg−1) which was at par with DOS-4 (940 g kg−1). In earlier investigations, methyl eugenol was reported as the major constituent of holy basil herb, leaf, stem and inflorescence (725 g kg−1, 753 g kg−1, 837 g kg−1 and 652 g kg−1) in respective concentration (55 g kg−1, 64 g kg−1, 27 g kg−1 and 12 g kg−1) in each oil (Kothari et al., 2005). The morphologically distinct ‘Rama’ and ‘Shyama’ types from India and many chemotypes within them have been reported to contain eugenol and methyl eugenol (Archana et al., 2013). As compared to the highest average eugenol share (913 g kg−1) in DOS-1 accession in our study, the earlier reported highest share of eugenol in clove basil under Uttarakhand conditions was only 775 g kg−1 (Sethi et al., 2013). The maximum Azuline was found in Dadiya P-1 (110 g kg−1) followed by Dadiya SP-1 (95 g kg−1) but was absent in DOS-1. Similarly, maximum share of β-Caryophyllene was observed in CHES G-1 and Dedia G-1 (182 g kg−1), but nil in DOS-1 and Angana. β-Caryophyllene was the second most dominant constituent from whole herb, leaf, stem and inflorescence in respective concentration (55 g kg−1, 64 g kg−1, 27 g kg−1 and 120 g kg−1) in 703
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Fig. 2. Relationship between no. of peltate gland (PG) and age of leaf.
each oil (Kothari et al., 2005). Out of the 11 accessions, Alfa-cubebene and delta-3-carene were absent in five accessions, namely, DOS-1, DOS3, CHES G-1, Dedia G-1 and Dadiya SP-1. In the remaining six accessions, their corresponding concentration was highest in Angana (124 g kg−1 and 27 g kg−1) followed by DOS-7 (35 g kg−1 and 5 g kg−1). Eugenol was the most dominant constituent followed by βCaryophyllene in the tested accessions. With the advancement of harvesting stages, the maximum share of eugenol elements at the onset of flowering, full flowering and crop maturity was 860 g kg−1, 890 g kg−1 and 990 g kg−1 in DOS-1 (increasing trend) while minimum share was observed in Angana (160 g kg−1, 580 g kg−1 and 770 g kg−1). However, reverse trend was observed for β-Caryophyllene in DOS-1 (100 g kg−1, 90 g kg−1 and 00 g kg−1) and Angana (200 g kg−1, 100 g kg−1 and 00 g kg−1), respectively (Figs. 4 and 5). The major oil constituents of O. tenuiflorum grown in Australia (methyl chavicol 870 g kg−1), Bangladesh (eugenol 417 g kg−1 and sesquiterpene components 459 g kg−1 in green type and eugenol 775 g kg−1 in purple type), Cuba (eugenol 343 g kg−1, β-elemene 180 g kg−1 and β-caryophyllene 231 g kg−1), Germany (eugenol 242 g kg−1, α-bisabolene 106 g kg−1, β-bisabolene 154 g kg−1 and methyl chavicol 116 g kg−1) and India (eugenol 350–530 g kg−1) are distinctly different from the chemotype reported in this study (Laakso et al., 1990; Pino et al., 1998; Raju et al., 1999). In earlier study, the level of eugenol and methyl eugenol in holy basil declined with progressive maturation of the plant leaves (Dey and Choudhuri, 1983). Eugenol, β-caryophyllene, E-methyl cinnamate and (trans)-β-guaiene were the most abundant components identified from three accessions (Sims et al., 2014). Influence of planting date and harvesting was recorded in three O. tenuiflorum accessions in Alabama. In accession PI 652056, the level of eugenol increased with a delay in harvest time, while, accession PI 652057, the level of β-caryophyllene was high at the 30-day harvest, but decreased significantly by the time of the 60-day harvest when eugenol became the dominant essential oil constituent (Sims et al., 2014). 3.3. Economics
Fig. 3. The no. of Peltate Glands (PG) per leaf at different leaf maturity stage.
Clove, is a major source of eugenol in India but is not produced in 704
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Table 5 Chemotypic composition of essential oils (g kg−1) at crop maturity stage in eleven accessions of holy basil. Accession
Eugenol element
Azuline
β-Caryophyllene
α-Cubebene
Delta 3 carene
α-humulene
TC-1 DOS-1 DOS-3 DOS-4 DOS-5 DOS-7 CHES G-1 Dediya G-1 Dediya SP-1 Dediya P-1 Angana
730j 990a 780g 940b 730j 880c 800e 800d 780h 790f 770i
39de 0.0 87bc 38de 89bc 64cd 3ef 4ef 95b 110a 5.0ef
119d 0.0 117e 0.0 139c 5.0h 182a 182b 116f 75g 0.0
13d 0.0 0.0 3.0e 22c 35b 0.0 0.0 0.0 2.0ef 124a
3.0c 0.0 0.0 4.0c 2.0d 5.0b 0.0 0.0 0.0 1.0d 27a
0.0 5.0c 6.0bc 0.0 2.0f 4.0d 8.0ab 9.0a 5.0c 2.0f 3.0e
Means with the same letter (superscript) in the columns do not showing significantly different (P = 0.05) − (Duncan Multiple Range Test). Fig. 4. Relationship between eugenol element and βCaryophyllene at different leaf harvesting stage.
Fig. 5. Chromatogram of volatile compound identified in accession (A) DOS-1 at onset of flowering; (B) DOS-1 at full flowering; (C) DOS-1 at maturity; (D) Angana at onset of flowering; (E) Angana at full flowering and (F) Angana at maturity from GC/MS.
necessitates the need to find out some cheaper alternatives like Ocimum species. In this study, basil accession DOS-1 has been found to be rich in eugenol content. On an average, a farmer can get gross returns from dry leaf and essential oil from fresh herb marketing approximately US$
sufficient quantity to meet the requirement of the country. Eugenol is usually extracted from the clove buds (70–85%) as well as from the leaves and barks of Cinnamomum (20–50%) (Shasany, 2016). Further, the high cost involved in the extraction of eugenol from clove, 705
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Fig. 6. Economics of Accession DOS-1 for leaf and oil yield/hectare.
Fig. 7. Field view of DOS-1 at farmer’s field as a Front-Line Demonstration.
4281 ha−1 and US$ 2838 ha−1 and B:C ratio over total cost was 1.6 and 2.2, respectively (Fig. 6). Compared with clove, Ocimum species can be grown in varied soil and climatic conditions. The crop is ready for harvesting in 90–95 days after planting and subsequent harvests can be taken in 60–65 days interval (Fig. 7). Besides, cost of production of different Ocimum species is far less compared with clove (Smitha and Tripathy, 2016). The holy basil is cheaper source for commercial extraction of eugenol (Mukherji, 1987). It is thus concluded that the holy basil accession DOS-1 can be commercially cultivated as potential sources of eugenol with good returns.
herbal medicines and other use applications. Competing interests The authors declare that they have no competing interest. Authors’ contribution Dr. Parmeshwar Lal Saran was involved in conception, design of the experiments and acquisition of data. He has collected all data and extracted the essential oil from the plants. Dr. Vandana Tripathy analyzed the essential oil by GCMS for the first two stages and Dr. Ajoy Saha for last maturity stage. Dr. Kalariya has analyzed chlorophyll and carotenoids content and Dr. Manish Kumar Suthar counted PG in all the accessions. Dr. Jitendra Kumar, Director, provided all the facilities and guidance.
4. Conclusion Different harvesting stages of O. sanctum accessions grown in western India have been investigated for desirable morphological and chemotypic variations. Maximum oil per cent in green herbage, dry leaf recovery, total chlorophyll, essential oil, carotenoids and eugenol elements was observed in DOS-1 accession at crop maturity. Similarly, the DOS-1 leaves contained maximum no. of peltate glands (PG) at all three stages. The maximum share of eugenol/methyl eugenol was observed in DOS-1 at onset of flowering, full flowering and crop maturity in increasing order with advancement of harvesting stages, while reverse trend was observed for β-Caryophyllene. Overall, holy basil and in particular DOS-1 accession was found superior and thus, can be considered for further improvement. Instead of costly clove, O. sanctum (basil) leaves offer cost effective substitute for herbal tea industry,
References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry. Allured Publishing Corporation, Carol Stream, Illinois. Anbarasu, K., Vijayalakshmi, G., 2007. Improved shelf life of protein-rich tofu using Ocimum sanctum (tulsi) extracts to benefit Indian rural population. J. Food Sci. 72, 300–305. Archana, P.R., Ashok, K., Dutta, M., 2013. Chemical characterization of aroma compounds in essential oil isolated from Holy Basil (Ocimum tenuiflorum L.) grown in India. Genet. Resour. Crop Evol. 60, 1727–1735. Dey, B.B., Choudhuri, M.A., 1983. Effect of leaf development stage on changes in essential oil of Ocimum sanctum L. Biochem. Physiol. Pflazen 178, 331–335.
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constituents of the leaves of Ocimum sanctum L. J. Essent. Oil Res. 11, 159–161. SAS, 2008. SAS Institute Inc. 2008. SAS/STAT® 9.2 User’s Guide. SAS Institute Inc, Cary, NC. https://support.sas.com/documentation/cdl/en/statugstatmodel/61751/PDF/ default/statugstatmodel.pdf. Saran, P.L., Tripathy, V., Meena, R.P., Kumar, J., Vasara, R.P., 2017. Chemotypic characterization and development of morphological markers in Ocimum basilicum L. germplasm. Sci. Hortic. 215, 164–171. Sethi, S., Praksha Om Chandra, M., Punetha, H., Pant, A.K., 2013. Antifungal activity of essential oils of some ocimum species collected from different locations of Uttarakhand. Indian J. Nat. Prod. Res. 4 (4), 392–397. Shasany, A.K., 2016. The holy basil (Ocimum sanctum L.) and its genome. Indian J. Hist. Sci. 51 (2), 343–350. Sims, C.A., Juliani, H.R., Mentreddy, S.R., Simon, J.E., 2014. Essential oils in holy basil (Ocimum tenuiflorum L.) as influenced by planting dates and harvest times in North Alabama. J. Med. Active Plants 2 (3), 33–41. Singh, S.K., Anand, A., Verma, S.K., Siddiqui, M.A., Mathur, A., Saklani, S., 2011. Analysis of phytochemical and antioxidant potential of Ocimum kilimandscharicum Linn. Int. Curr. Pharma Res. J. 3, 40–46. Smitha, G.R., Tripathy, V., 2016. Seasonal variation in the essential oils extracted from leaves and inflorescence of different Ocimum species grown in Western plains of India. Ind. Crops Prod. 94, 52–64. Szabo, K., Nemeth, E., Prapzha, L., Bernath, J., Pank, F., 1996. Morphological and chemical variability of basil genatypes. International Symposium on Breeding Research on Medicinal and Aromatic Plants Quedlinbury Germany vol. 2 (1), pp. 76–79. Tan, K.H., Nishida, R., 2012. Methyl eugenol: its occurrence, distribution, and role in nature, especially in relation to insect behavior and pollination. J. Insect Sci. 12, 56 (insectscience.org/12.56). Tilwari, A., Tamrakar, K., Sharma, R., 2013. Use of random amplified polymorphic DNA (RAPD) for assessing genetic diversity of Ocimum sanctum (Krishna tulsi) from different environments of Central India. J. Med. Plants Res. 7 (24), 1800–1808. Turner, G.W., Gershenzon, J., Croteau, R.B., 2000. Development of peltate glandular trichomes of peppermint. Plant Physol. 124, 665–679. Wagner, G.J., 1991. Secreting glandular trichomes: more than just hairs. Plant Physiol. 96, 675–679.
Gang, D.R., Wang, J., Dudareva, N., Nam, K.H., Simon, J.E., Lewinsohn, E., Pichersky, E., 2001. An investigation of the storage and biosynthesis of phenylpropenes in sweet basil. Plant Physiol. 125 (2), 539–555. Guo, J., Yuan, Y., Liu, Z., Zhu, J., 2013. Development and structure of internal glands and external glandular trichomes in Pogostemon cablin. PLoS One 8 (10), e77862. http:// dx.doi.org/10.1371/journal.pone.0077862. Herrmann, K.M., Weaver, L.M., 1999. The shikimate pathway. Ann. Rev. Plant Physiol. Plant Mol. Biol. 50, 473–503. Kothari, S.K., Bhattacharya, A.K., Ramesh, S., 2004. Essential oil yield and quality of methyl eugenol rich Ocimum tenuiflorum L.f. (syn Ocimum sanctum L.) grown in south India as influenced by method of harvest. J. Chromatogr. A 1054, 67–72. Kothari, S.K., Bhattacharya, A.K., Ramesh, S., Garg, S.N., Khanuja, S.P.S., 2005. Volatile constituents in oil from different plant parts of methyl eugenol-rich Ocimum tenuiflorum L.f. syn. O. sanctum L.) grown in South India. J. Essent. Oil Res. 17 (6), 656–658. Laakso, L., Seppanen-Laakso, T., Hermann-Wolf, B., Knobloch, K., 1990. Constituents of the essential oil from the Holy basil or Tulsi basil, Ocimum sanctum. Planta Med. 56, 527. Malav, P., Pandey, A., Bhatt, K.C., Gopala Krishnan, S., Bisht, I.S., 2015. Morphological variability in holy basil (Ocimum tenuiflorum L.) from India. Genet. Resour. Crop. Evol. 62, 1245–1256. Mukherjee, P.K., Maiti, K., Mukherjee, K., Houghton, P.J., 2006. Leads from Indian medicinal plants with hypoglycemic potentials. J. Ethnopharmacol. 106, 1–28. Mukherji, S.P., 1987. Ocimum-a cheap source of Eugenol. Science Reporter, p. 599. 31. Eugenol-rich Ocimum variety released. CSIR NEWS 45. PID CSIR, New Delhi, pp. 256. NIST, 2005. Mass Spectral Library. NIST Mass Spectral Search Program (NIST 05, Version 2.0d). The NIST Mass Spectrometry Data Center, Gaithersburg, MD. Padalia, R.C., Verma, R.S., 2011. Comparative volatile oil composition of four Ocimum species from northern India. Nat. Prod. Lett. 25, 569–575. Pino, J.A., Rosado, A., Rodriguez, M., Garcia, D., 1998. Composition of the essential oil of Ocimum tenuifl orum L. grown in Cuba. J. Essent. Oil Res. 10, 437–438. Pistrick, K., 2001. Ocimum. In: In: Hanelt, P., Institute of Plant Genetics and Crop Plant Research (Eds.), Mansfeld́ s Encyclopedia of Agricultural and Horticultural Crops, vol. 4. Springer, pp. 1954–1960. Raju, P.M., Ali, M., Velasco-Negueruela, A., Perez-Alonso, M.J., 1999. Volatile
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