Pre-Flowering Harvesting of Ocimum gratissimum for Higher Essential Oil and Eugenol Yields Under Semi-Arid Tropics
Kothari, Sushil K
In spice basil (Ocimum gratissimum L.), contribution of leaves, stalks and inflorescences were 59%, 23% and 18% to total plant biomass and 80%, 1% and 19% to total oil yield, respectively. The leaf oil was richer in eugenol but simultaneously had lower (E)-β-ocimene, compared to the inflorescence oil. Harvesting at pre-flowering produced 12.5%, 24.1%, 35.5% and 50.0% higher biomass yield compared to harvesting at 25%, 50%, 75% and 100% flowering, respectively, in the first year of cropping. The respective increase was 16.8%, 22.0%, 38.2% and 63.2% in the second year. Late harvested crop ( 100% flowering) contained the highest amount of essential oil and it decreased in the order of harvesting at 100% flowering > 75% flowering > 50% flowering > 25% flowering > pre-flowering treatment. The total oil yield was, however, significantly higher (15.8-19.9% and 12.7-33.6% in first and second years, respectively) with pre-flowering compared to all other harvest treatments. Pre-flowering harvested crop produced oil containing the highest amount of eugenol and it decreased in the order of harvesting at pre-flowering > 25% flowering > 50% flowering > 75% flowering > 100% flowering treatment.
Key Word Index
Ocimum gratissimum, Lamiaceae, harvest management, oil yield, essential oil composition, eugenol, (E)-β-ocimene.
The genus Ocimum (family: Lamiaceae), collectively called basil, consists of about 160 species (1), and is spread over the tropical, sub-tropical and wannerpart of the temperate regions of both the hemispheres ranging from sea level to 1800 ft altitude. Spice basil (O. gmtissimum L.), perennial and drought tolerant, is one of the important basil types and is cultivated in many Asian and Mediterranean countries and the United States. It is cultivated in about 2000 hectares in the northern and southern parts of India. Leaves and flowering tops of the plant yield an essential oil on steam distillation. The oil is used to flavor foods, dental and oral products, in fragrances and aromatherapy and in traditional rituals and medicines. Eugenol is the dominant constituent in the oil of O. grntissimum of the Indian (2-5), and Brazilian native plants (6,7) as against thymol and 1,8 cineole in Cameroon native plants (8), and thymol, γ-terpinene and pcymene in Togo and Benin native plants (9,10). Another rare chemotype rich in geraniol is also known to exist in East Africa (11). The oil of O. gratissimum of Indian and Brazilian origins being an important industrial source of eugenol, may replace pure eugenol in many pharmaceutical and cosmetic compositions.
Earlier, a large variability has been demonstrated in respect to composition of oil derived from leaf and inflorescence in O. tenuiflorum (12), O. basilicum (13), and O. micranthum (14). Similarly, changes in oil content and composition were observed with progressive stages of leaf development in O. tenuiflorum (15), plant height in O. basilicum (16), stage of crop harvest in O. basilicum (17), and O. gratissimum (17), and with season of harvest in O. gratissimum (4). There is, however, dearth of information on O. gratissimum in respect to yield and composition of oil obtained from different crop segments and as influenced by stage of crop harvest under semi-arid tropics.
Materials and Methods
Field experiments were conducted in 1999-2002 at the Research Farm of the Central Institute of Medicinal and Aromatic Plants, Resource Centre, Hyderabad (542 in above msl, 17°20′ N and 78°3′ E), India. The annual rainfall is about 760 mm of which 80% is received between June and September (southwest monsoon). The average temperature is 29°C, and varies from 22°-35°C, the highest (44°C day temperature) being in May and the lowest (12°C night temperature) in January. The winter season is characterized by mild, cool dry weather. The experimental location has a semi-arid tropical climate.
The soil of the experimental site was well drained, red sandyloam (alfic ustochrept) in texture, having organic carbon 0.3%, pH 7.3 and available N, P and K at 60.3, 9.5 and 142.5 kg/ha, respectively. Ocimurn gratissimum seeds were collected from cultivated fields in South India and six week old seedlings were transplanted in 10 x 20 m size plot in July 1999. Before final land preparation, neem cake (0.51 ha^sup -1^), phosphorus (40 kg P^sub 2^O^sub 5^/ha) and potassium (40 kg K^sub 2^O/ha) were applied and mixed well with the top soil of plot area. Nitrogen (60 kg N/ha) was applied into two equal split doses at 30 and 45 days after transplanting (DAT). At full bloom stage (77 DAT), observations were recorded on plant height and spread, number of branches and leaves/plant, leaf color (dorsal and ventral surface), shape, margin, length, width and size, inflorescence number/plant, color, diameter and length and flower color and diameter. On five randomly selected plants, apical part (25-35 cm) of all branches, including inflorescence, was harvested and whole herb weight/plant was recorded. Data were also recorded in respect of biomass distribution in leaf, stem and inflorescence. The oil content in leaf, stalk and inflorescence were separately determined along with whole herb (each three replications of 100 g each) following hydrodistillation in Clevenger-type apparatus for 4 h.
The oil was analyzed using a Perkin Elmer gas chromatograph (Model 8500) equipped with FID and a capillary column BP-I (25 m x 0.5 mm, 0.25 μm thickness) coated with dimethyl siloxane. Nitrogen was used as carrier gas at a 10 psi inlet pressure. Temperature programming was 60°0 -220°C at a ramp rate of 5°C/min with a final hold time of 10 min. Samples (0.1-0.2 μL) were injected neat with a split ratio 1:80. GC/MS analysis was done on a Shimadzu (Model QP-2000) equipped with a capillary column OV-1 (50 m x 0.25 mm, 0.25 μm thickness). Carrier gas used was helium at a flow rate 2 mL/min with temperature programming 100°C (6 min) 100°-250°C at 10°C/min. The compounds were identified by comparing their relative retention indices of the peaks with those of standard compounds under the same conditions, peakenrichment on co-injection with authentic samples, GC/MS and with the literature data (20,21). Quantitative data was obtained by electronic integration of peak areas (FID) without the use of response correction factors.
Second field experiment was initiated in June 2000 in the same block of the research farm. The experiment was laid out in a randomized block design with five treatments on stage of crop harvest (pre-flowering and 25%, 50%, 75% and 100% flowering) and four replications, individual plots being 3 x 6 m. Each plot received uniform dose of neem cake 900g(0.5t/ha),di-ammonium phosphate 155 g (40 Kg P^sub 2^O^sub 5^/ha) and muriate of potash 120 g (40 kg K^sub 2^O/ha) as basal dose which was incorporated with 5 cm top soil using hand hoe. Ocimum gratissimum seedlings, six weeks old, were planted at 60 cm row-to-row and 45 cm plant-to-plant spacing in June 2000. The field was irrigated immediately after planting for early establishment of the seedlings. Thereafter, the field was irrigated 11 and 13 times in the first and second year of experimentation, respectively. Nitrogen at 120 kg/ha was applied in the form of urea spreading over all the harvests per annum. The crop received five and four hand weedings during first and second year of experimentation. Apical part (25-35 cm) of all the branches was harvested in all the treatments as given below:
Biomass yield was recorded plot-wise at each harvest and essential oil content in biomass was determined and the GC analysis of the oil samples from first harvest were performed as stated earlier.
All the data from second experiment were subjected to statistical analysis of variance (ANOVA) technique as applicable to randomized block design (19).
Results and Discussion
Ocimum gratissimum cv. Indian is perennial in growth habit with moderate plant height 76.5 ±5.1 cm and spread 58.1 ± 6.3 cm, number of branches/plant 14.5 ± 3.0, leaf color green (ventral surface) to dark green (dorsal surface), shape ovate, margin crenate serrated, size 48.3 ± 17.7 cm^sup 2^ and number/plant 670 ± 51, inflorescence color greenish with violet tinge, length 17.1 ± 2.8cm, diameter 1.65 ± 0.15 cm and number/plant 90.7 ± 8.9 and flower color white and diameter 2.5 ± 0.5mm. On an average, contribution of leaves, stalks and inflorescences were 59%, 23% and 18% to total plant biomass and 80%, 1% and 19% to the total oil yield, respectively (Table I). Oil content in stalk being very low, its contribution to total oil yield was negligible. On the contrary, leaf contribution to total oil yield was very high because of higher leaf biomass and oil content. A marked variability was observed in respect of composition of the oils from leaves and inflorescences (Table II). The leaf oil was richer in eugenol but simultaneously had lower (E)-β-ocimene, as compared to inflorescence oil. Remarkable variability in volatile constituents from leaves and inflorescence oil of O. tenuiflorum (12), O. micranthum (14), O. basilicum (13), and Cameroon-type O. gratissimum (8), has also been reported. The oil distilled from whole herb contained eugenol as the dominant compound which corresponds to earlier reports on oil composition of O. gratissimum grown in India (3,4).
Harvesting of O. gratissimum at pre-flowering gave maximum biomass yield during both the first and second year of experimentation (Table III). The pre-flowering treatment produced 12.5%, 24.1%, 35.5% and 50.0% higher biomass yield compared to 25%, 50%, 75% and 100% flowering, respectively, in the first year. Likewise, the respective increase was 16.8%, 22.0%, 38.2% and 63.2% in the second year. This is attributed to frequent and higher number of harvest in the pre-flowering treatment. On the contrary, highest oil content was observed in the late harvested crop (100% flowering) during both the years of cropping. Oil content in different treatments was in the order of 100% flowering > 75% flowering > 50% flowering > 25% flowering > pre-flowering. Likewise, a maximum of 0.4% essential oil content on fresh weight basis was earlier observed in O. gratissimum at full bloom stage (3). Maximum oil yield was, however, observed in the pre-flowering treatment as that of biomass yield during both the years. The pre-flowering treatment gave 16.7%, 15.8%, 19.3% and 19.9% higher oil yield compared to 25%, 50%, 75% and 100% flowering treatment, respectively, in the first year. The respective increase was 13.4%, 12.7%, 13.1% and 33.6% in the second year. Maximum oil yield of O. gratissimum was earlier observed in northeast India with four harvests in a year at 90 days interval (4). Like oil yield, remarkable treatment differences were observed in respect of oil composition. Maximum eugenol content in oil was observed in the pre-flowering treatment as that of biomass and oil yields (Table IV). Eugenol content in different treatments was in the order of pre-flowering > 25% flowering > 50% flowering > 75% flowering > 100% flowering. Earlier decline in major oil constituents like eugenol and methyl eugenol in O. tenuiflorum oil was observed with progressive maturation of leaf (15). In O. gratissimum seasonal variation in composition of essential oil is relatively well known (4), compared to stage of crop harvest. Co-incidence of maximum oil yield and eugenol content in O. gratissimum oil in the pre-flowering treatment, suggests a high significance of stage of crop harvest for getting higher oil and eugenol yields. Unlike eugenol, (E)-β-ocimene, the second major oil constituent, declined in the reverse order such that the lowest content was observed in the pre-flowering treatment. Little variability was observed among the treatments with respect to the 27 other minor and trace constituents of oil.
The results show that leaf contributed about 80% to total oil yield of O. gratissimum. The leaf oil was richer in eugenol compared to inflorescence oil. Further harvesting at pre-flowering stage has definite advantages under semi-arid tropics because of highest oil yield together with superior quality on account of higher eugenol content in oil.
The authors thank the director, CIMAP, Lucknow for providing necessary facilities.
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Sushil K. Kothari,* Arun K. Bhattacharya and Kamla Singh
Central Institute of Medicinal and Aromatic Plants (CIMAP) Renounce Centre, Boduppal, Uppal (PO), Hyderabad-500 039, India
Srinivas I. Ramesh and Eranki V.S. Prakasa Rao
CIMAP, Field Station, GKVK (PO), Allalasandra, Bangalore-560 065, India
CIMAP, P.O. CIMAP, Lucknow – 226 015, India
* Address for correspondence
Received: December 2002
Revised: February 2003
Accepted: April 2003
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