INTRODUCTION:

Man has evolved the usage of nature for the treatment and prevention from the diseases. Plants, animal and minerals are being explored for their beneficial effects. Due to the large number, easily available and reproducible character plants gather much more attention as the therapeutic agent against various disorders/diseases. Earlier, the plants were characterized on the basis of their morphological characters but scientific studies have showed that similar morphological plant also differs in their therapeutic potency. The plant kingdom includes Lamiaceae (Labiatae) family, which is also known as the Mint Family comprising of 236 genus including Ocimum, well-known as Basil¹. Different species of Ocimum is found in different regions of the world have different chemical composition with significant therapeutic values and clinical efficacy. The species of Ocimum have also been regarded as the “King of Herbs” due to its enormous medicinal importance². One of the species of Ocimum was being known to be originated from the hills of Kilimandscharo of Africa and was being named Ocimum kilimandscharicum³. Previously, an attempt has been made to compile the phtyochemistry, pharmacology and traditional potency of the plant^4,-6. In the present article the pharmacognostic aspects of this ethnomedicinal plant have been collectively described including its synonyms, common names, allied species, taxonomical classification, geographical distribution, cultivation, collection, morphology and microscopy which will help to identify that the similar name or properties should not to be confused with the authentic source. In-depth study of phytochemicals illustrates the presence of reported volatile as well as non volatile components along with the factors effecting variation in yield and components. Apart from traditional uses and pharmacological activities, this manuscript draws the attention towards the current economic, environmental and social benefit describing the practicality of the efforts involved in the transition of ordinary plant into an immense important plant.

SYNONYM:

As per the literature and information available, some of the species has been regarded as the synonym of O. kilimandscharicum like O. camphora Guerke, O. johnstonii Baker, O. tortuosum Baker^7,8.

COMMON OR VERNACULAR NAMES:

The ‘Camphor Basil’ is used as the common trade name for O. kilimandscharicum due to the presence of camphor and widely acceptable of English language all over the world⁹. It is native to Africa, but due to the distribution of the plant throughout the world, it is being expressed differently in different languages. The common or vernacular names of the plant in local language of the different nations are being described in (Table 1) along with references.

Table 1: Common or Vernacular Names of O. kilimandscharicum

S. No.	Common or Vernacular Name	Language (Country)
1.	Alfavaquinha¹⁰	Portuguese (Brazil)
2.	Basilicao-canforado¹¹	Portuguese (Portugal)
3.	Camphor Basil^{7, 12, 13,14}	English (All over the World)
4.	Fever Plant^{15, 16}	Swahili (Kenya)
5.	Gakuki¹⁷	Kikuyu (Kenya)
6.	Gethereti¹⁸	Meru (Kenya)
7.	Hoary Basil¹⁵	Swahili (Kenya)
8.	Kampferbasilikum¹¹	German (Germany)
9.	Kapoor Tulsi^{12, 13, 14, 19}	Hindi (India)
10.	Kapur Tulsi^{12, 13, 14, 19}	Hindi (India)
11.	Karppura Tulasi¹²	Malyalam (India)
12.	Karpur Tulsi^13,14,19	Bengali (India)
13.	Karpura Tulasi¹²	Sanskrit, Kannada, Tamil, Telugu (India)
14.	Kilimanjaro Basil¹⁵	Swahili (Kenya)
15.	Lisuranza¹⁸	Luhya (Kenya)
16.	Makori¹⁸	Meru (Kenya)
17.	Mbirirwa¹⁸	Marakwet (Kenya)
18.	Mutei¹⁸	Kikuyu (Kenya)
19.	Mwonyi¹⁸	Luhya (Kenya)
20.	Rehan²⁰	Arabic (Yemen)
21.	Ufagio²¹	Kiswahili (Tanzania)

ALLIED SPECIES:

Leaves of Artemisia nilagirica have camphor like smell, due to which it is used as Karpuratulasi in Kerala (India). The same plant in Malyalam (India) is known as Makkippu, but is not mentioned as Karpuratulasi in any of the literature available¹². Diverse geographical source has evolved genetic diversity in Ocimum species²². Another species found in Kamba with local name Mukandu munini, was identified as Ocimum pseudokilimandscharicum. The powder of the leaves is used to cure stomachache when taken with water¹⁸. O. pseudokilimandscharicum is regarded one of the sub- sub species of the variety gratissimum. This variety has hairy stem all over with pubescent leaves²³. In accordance to the literature, it is also called as African blue basil^2,24. During the survey, it is revealed that a hybrid formed by the crossing over of O. kilimandscharicum and O. basilicum is known as African blue basil or Dark Opal which is written as Ocimum kilimandscharicum X basilicum. It has purple color leaves with strong camphor aroma^25,26.

TAXONOMIC CLASSIFICATION:

For the systematic study of the plants, scientific classification is adopted. The hierarchy (in decreasing order) of the plant kingdom includes division, class, order, family, genus, species, sub species and varieties²⁷. The official taxonomic classification of the kilimandscharicum species for the genus Ocimum is as follows:

Kingdom :Plantae – Plants

Subkingdom :Tracheobionta – Vascular plants

Superdivision :Spermatophyta – Seed plants

Division :Magnoliophyta – Flowering plants

Class :Magnoliopsida – Dicotyledons

Subclass :Asteridae

Order :Lamiales

Family :Lamiaceae ⁄ Labiatae – Mint family

Genus :Ocimum L. – basil

Species :Ocimum kilimandscharicum Guerke – hoary basil

GEOGRAPHICAL DISTRIBUTION:

It is native of East tropical countries of Africa like Kenya, Tanzania and Uganda^11,28,29. It is cultivated on a commercial scale in central west part of Africa continent³⁰. The International Centre of Insect Physiology and Ecology (ICIPE), with the support of Muliru Farmers Conservation Group (MFCG), Biovision Trust of Africa University of Nairobi, World Agroforestry Centre and Kenya Forestry Research Institute, helped the Muliru farmers to build an alternate source of income by domestication and commercialization of the medicinal plant in Kakamega Forest situated in western Kenya³¹. During the Second World War, it was being distributed all over the world as a source of camphor. The attempts were done in different countries for the cultivation of the plant and isolation of camphor³². Department of Agriculture of United States also initiated the development of cultural practice required for the cultivation of Ocimum kilimandscharicum by using seeds and seedling³³. During the 1953-54, cultivation was much emphasized in India as it was importing camphor and camphor oil from other countries like Japan and Hongkong³⁴. Experimental cultivation was undertaken in various areas and encouraging results were reported from various states of India like Uttar Pradesh, West Bengal, Maharashtra, Mysore, Kerala, and Jammu¹³. Later, it is being cultivated in various parts of India like West Bengal, Assam, Tamil Nadu, Karnataka, and Kerala^14,19,35. Although, cultivation was tried in different parts of India but the native distribution of the plant also appeared to be in the peninsular India as it is found growing wildly in Kapilash wildlife sanctuary, Dhenkanal district and Forest near Begunia of Khurda district, Odisha, which has not reported so far³⁶. In another study, this herbaceous plant is also being reported to be native of Brazil¹⁰. Across the world, the plant is also distributed in various countries like Bolivia, Caribbean, China, French Guiana, Gabon, Guyana, Madagascar, Mexico, Panama, Suriname, Turkey, United States and Venezuela^10,15.

CULTIVATION AND COLLECTION:

The detailed agronomical aspects like establishing seed bed nursery, seed selection and preparation, site selection, land preparation, sowing of seeds in seedbed, transplanting, protection, pest control, irrigation, harvesting, post harvesting handling, processing requirements are described in details based upon the experiences of farmers cultivating and processing in various parts of Africa. The experimentation on cultivation in India and other parts of the world has also been provided many attribute for the better yield. Combination of these experiences could be regarded as “Good Agricultural Practices” for the cultivation of O. kilimandscharicum.

Propagation:

The growing of a new plant from any part of plant or propagation is done usually by two methods. In the seed propagation 70-140gram properly hygienically stored seeds are sufficient for producing the crop in 1 hectare land. Due to the small size, the seeds are mixed with dried powdered soil and then the mixture is placed at the distance of 150mm in shallow furrow after the middle of March in nursery bed. The seeds may also be distributed evenly in thin layers and covered by fine layer of soil. Broadcasting method has also been successfully employed for the sowing of the seeds. Two ounces seeds are necessary for the seedlings required for 1 acre of land^15,34,37. The seed bed may be covered by grass or banana leaves to protect from bird and direct sunlight. Seeds take about 1-3 weeks for the germination. The rate of germination from seeds is about 70-80 %³⁸. After the germination, seedlings of 5-7 weeks could be transplanted in field. Seed propagation is most suitable method for the farmers^15,24. In the cutting propagation, cutting are taken from the healthy and fully grown plant is planted in nursery bed. Cutting began to sprout in 7-10 days and ready for transplantation in 4-5 weeks. High leaf yield is obtained by this method^13,15.

Soil and Irrigation:

It can be grown on a wide variety of soils under irrigated and non irrigated conditions but grows well in clayey or sandy soil at an annual rainfall of 1250cm or good irrigation facility. The nursery bed is prepared by the breaking of soil compaction (primary tillage) followed by removing all kinds of waste material (secondary tillage). Evening time is preferred for the transplantation. The flowering of the plant begins after 14 weeks of germination and at this stage the height of the plant is approximately 15 inches^13,15. The plant gives more shoot after every cutting, so the appropriate space between two plants should be 2 feet x 2 feet or 11,000 plants per hectare³³. Occasional weeding is done manually by removing the unwanted plants³⁴.

Harvesting:

The plant is ready for the harvesting after 4-6 months of transplanting. The plant can be harvested for three times per annum for 3 to 5 years. The first harvesting is done when the plant eight reaches 60cm height just the beginning of flowering. The harvesting is done mainly in the months of August to September, November- December and March-April. In the cooler climate the harvesting is done only twice a year^13,15.

Collection and Storage:

The plants are cut 50-75mm above the ground and collected in heaps. The leaves are removed and dried in air for later use. Approximately 2,200-5500kg per hectare dried leaves could be cultivated but also depends upon various geographical, climatic and agricultural conditions. The plant thrives perennial and no protection is required as it is also not grazed by the animals^13,15.

Miscellaneous Factors:

Although no manuring is required, but the farmyard manure, compost, manure mixture or Ammonium sulphate is used before flowering stage. Usage of high manure and chemicals have shown good herbage yield but low oil content^13,15. The plant is also susceptible to termites, attacking to the roots of the plant after the first harvesting. The effect could be deterred by sprinkling alkaline ash around the seedbed furrows¹⁵. Significant reduction in the plant yield and essential oil was noted when soil of three weeks old seedlings were inoculated with larvae of nematode Meloidogyne incognita. The Chemical pesticide (Aldicarb) and Organic Pesticide (Neem cake) usage shows the promising result by inhibiting the population of nematode³⁹. The vegetative growth was found to be slower at an altitude above 1000 feet and experimentation in Srinagar which is situated 5000 feet above the ground proved the zero survival rates in frost³⁷. On long storage the seeds were also reported for losing their viability³⁴.

MACROSCOPICAL CHARACTERISTICS:

Due to much higher polymorphism and cross pollination in the Ocimum genus, various types of species along with subspecies and varieties make the difficulty in maintaining appropriate botanical nomenclature. Although, some scientist have divided the Ocimum genus into two major species group i.e. Basicilicum and Sanctum. Basilicum group is characterized by petiolate bracts, conspicuous flower and black and ellipsoidal seeds, which become mucilaginous in water. The Sanctum group is characterized by sessile bracts, less conspicuous flower, brownish and ellipsoidal to globose seeds, which do not become mucilaginous in water⁴⁰. The morphological, chemical and genetic variability studies among the different species of consider O. kilimandscharicum as a variant species of basilicum⁴¹. The morphology, quantification of size and weight of plant and plant parts through principal component analysis among the various species of Ocimum showed clear and unambiguous identification of O. kilimandscharicum as tall plant, large gray–green inflorescence with camphor’s smell⁴². It is an aromatic and perennial under shrub with erect branchlet and can attain maximum height of 2 meters. The morphological characteristics of different parts of plant reported by various researchers^12,23,36 have been described collectively.

Leaves:

Leaves are simple, dorsiventral, petiolate, oppositely decussate arranged, elliptic, ovate or lanceolate shaped, acute to cuneate base, acute apex, serrate or deeply serrated margin, green to pale green color, strong, pungent, aromatic or camphoraceous odour with slight bitter taste. The presence of fascicular young leaves is also reported which may be mystified with stipules at the base. Size of leaf varies from 1.5 - 6cm long and 1 - 3 cm broad while petiole is 0.4 – 1cm long. The presence of indumentums over leaf and petiole makes the surface glandular, pubescent or hairy.

Stem:

The new stem is green which becomes woody in nature and acquire brownish color. The presence of white colored antrose glandular hairs with numerous sessile glands is common in newly formed stem. The loss of hairs starts from the base as it starts converting into woody stem. The peeling of epidermal layer makes the bark shredded. It is round to quadrangular shaped, highly branched which becomes denser on inflorescence axis.

Inflorescence:

The inflorescence is vertical, racemose, greenish-grey color, composed of 4-6 flowered whorls are attached at 2-15mm apart. The size of the inflorescence axis or spike varies from 14-30cm long.

Bracts:

Bracts are ovate shaped, curved (like making a comma), entire margin, acuminate apex, attenuate base, cuspidate apex, covered with hairs, length 2-3.5mm and width 1-2 mm in size.

Flowers:

Small and dense flowers with pedicel of 2-3mm long present in hairy spike are attached in a manner showing the reduction in distance of the flower whorl towards the apex.

Calyx:

The calyx is downward pointing, campanulate shaped, 2-5mm long during anthesis, 3–6mm long during fruiting and bi-lipped. The posterior lip is broad, rounded, accrescent, with decurrent base, mucronate reflexed apex and numerous sessile glands near pedicel. The anterior lip is four toothed with open throat. The two median teeth are lanceolate, acuminate apex, curved upwards and size similar to posterior lip. The two lateral lips are cuspidate, shorter and have deltoid teeth. Throat base have much denser ring of villose hairs than posterior lip.

Corolla:

White to light purple color, funnel shaped, 7-9.5mm long (including tube) and bi-lipped. The upper or posterior lip is truncated shaped and tetra-fid. The two median apparently joined lobes are ovate oblong and slightly longer than two lateral lobes which are broader. The lower or anterior lip is elliptic oblong and comparatively whiter than the upper lip. The lobes are pubescent dorsally.

Stamens:

Total of four stamens are present in two pairs, posterior, exserted, declinate, 3-5 mm long with the presence of transverse hairy projections near the base. Anther cells are confluent while filaments are free.

Pistil:

Ovaries are glabrous. Style is thin and bifid.

Seed:

Seeds of size 1-1.3 mm in diameter are black colored, pitted with ridge and scanty mucilage layer.

Fruits:

Fruits are like nutlets of brownish black color, smooth, ovoid- oblong shaped, minutely tuberculate and produce mucilage when soaked in water.

MICROSCOPICAL CHARACTERISTICS:

Transverse section of the leaf shows isobilateral lamina covered with cuticle, glandular trichomes with multicellular head and multi cellular warty covering trichomes. Mid rib is arc shaped vascular bundle consisting of xylem and phloem. Three to four layers of collenchymatous tissue present on upper side of vascular bundle. Powder microscopy shows multicellular and warty covering trichomes, diacytic stomata, vessels with spiral thickenings and fragment of lamina⁴³. The leaf bears diacytic stomata on adaxial as well as abaxial surface²⁴. Occasionally anomocytic stomata are also found. The quantitative microscopic parameters like frequency of stomata (416 per square meter), stomatal index (56.52), size of guard cell (27.52 X 6.88 µm) and size of pore (10.34 X 3.60 µm) were also reported⁴⁴. Scanning electron microscopy (SEM) studies revealed that shape of the seed is ellipsoid, broadened towards base, upper end rounded, lower end gradually tapered with distinct areola marks. Seed surface inconspicuously reticulate with raised walls, cells rectangular to polygonal, lumen floor shallow, sometimes with wavy uneven raised surface⁴⁵.

KARYOMORPHOLOGICAL PROPERTIES:

The detailed cytology and karyomorphology study of O. kilimandscharicum stated that the plants collected from two different places did not differ in morphological characters and have same chromosomes number i.e. 2n=76. The study revealed the difference in the size of the chromosomes. During evolution due to the change in structure with minor differences in nucleolar chromosomes have also been noted. 50.90 % pollen sterility at the lower pair of stamen was also observed which indicated the gradual sterilization of stamen. The species being an aneuploid also showed high degree of meiotic irregularities and multivalent formations^40,46,47.

PHYSIOCHEMICAL PROPERTIES:

The study of physiochemical properties have significant role in the preparation of standards for the crude drug material. The products derived from the plant parts also provide specific characteristic value that helps in detection of the adulterants. With the above point of view the various physiochemical parameters like moisture content, extractive value, ash value, refractive index, pH, acid value, acetyl value, ester value, saponification value, iodine value, optical rotation value and specific gravity value of the plant part and the derived product have been described in (Table 2).

Table 2: Physiochemical Properties of O. kilimandscharicum

Plant Part or Derived Products	Physiochemical Property	Value (Temperature, if any)
Aerial Parts⁴⁸	Moisture	80.55 %
	pH (10%)	6.04
	Water extract	24 %
	Alcoholic extract	5.6 %
	Ash Value	11 %
	Acid Soluble Ash	10.45 %
Essential Oil^37,38,49	Acetyl Value	55.3 to 118.1
	Acid Value	1.12 to 12.9
	Ester Value	5.07 to 27.5
	Optical Rotation	+ 33.7⁰(37⁰C)
	Optical Rotation	+27⁰36’ to 45⁰29’ (24⁰C to 34⁰C)
	Refractive Index	1.4716 (37⁰C)
		1.4606 (20⁰C)
		1.4610 to 1.4724 (24⁰C to 30⁰C)
	Specific Gravity	0.878 (15⁰C);
	Specific Gravity	0.9173 to 0.9846 (29.5⁰C to 33⁰C)
Decamphorised Essential Oil⁵⁰	Acid value	2.9
	Refractive index	34.7 (40⁰C)
	Saponification Value	17.02
	Specific Gravity	0.9190 (40⁰C)
Seeds (Dried)¹³	Ash	6.5 %
	Crude Fiber	27 %
	Moisture	6.4 %
Seed Oil^13,50	Yield	12.5 %
	Refractive Index	1.4852 (25⁰C) 1.4795 (40⁰C)
	Iodine Value	192.6 - 195.6
	Optical Rotation	1.4767 (40⁰C)
	Saponification Value	292 193

PHYTO-CHEMICAL CONSTITUENTS:

The major proportion of the phyto-constituents that exists in the plant are terpenoids in the form of essential or volatile oil. The highest quantity of essential oil was recorded in kilimandscharicum species when compared between different genotype of Ocimum species ⁵¹. The percentage yield of the essential oil and its component varies when isolated from the different geographical and climatic conditions. Maximum yield of oil 4.7% was reported from the plant collected from Kashmir, India³⁷. The percent oil of the flower is 1.80 % v/w while stem contains very minute quantity of oil³³. In majority of the cases, the monoterpenoids constitute the major proportion of volatile oil. The presence and percentage of the major constituents like Camphor, 1,8-Cineole, Camphene, Limonene, (E)-β-Ocimene, α-Pinene, Terpinen-4-ol and Terpinolene as illustrated in (Fig.1) also varies when collected and analyzed in different regions of the world^52-57. (R)-configuration dominates over the (S)-configuration during the investigation of enantiomeric chiral pairs viz., α-Pinene, Camphene, Limonene, Camphor, Linalool, Terpinen-4-ol, α-Terpineol and Germacrene D using 6-tertiarybutyldimethylsiliyl-2,3-diethyl- β-cyclodextrin except Borneol⁵⁸.

Fig. 1: Major Essential Oil Chemical Constituents of O. kilimandscharicum

The other monoterpenoids and sesquiterpenoids that have been reported in the essential oil are Aromadendrene, α-Bisabolene, β-Bisabolene, g-Bisabolene, Iso-Borneol, Bornyl Acetate, α-Bergamotene, β-Bourbonene, (E)-Caryophyllene, (Z)-Caryophyllene, Carveol, Carvone, Caryophyllene Oxide, Citronellol, α-Cubebene, Cubebol, Cubenol, ar-Curcumene, p-Cymen-8-ol, p-Cymene, α-Cadinol, Cadrenol, α-Campholenal, α-Copaene, β-Copaene, β-Cubenene, δ-Cadinene, Camphene Hydrate, δ-3 Carene, Chavicol, α-Elemene, β-Elemene, Ethylamyl carbinol, Eugenol, Methyl chavicol, Eudesmol, Elemol, (E)-β-Farnesene, (Z)-β-Farnesene, Fenchone, 2-epi-Funebrene, Bicyclogermacrene, Germacrene-D-4,ol, Geraniol, Geranyl acetate, Globulol, α-Gurjunene, α-Humulene, Humulene epoxide-II, cis-Linalool oxide, trans-Linalool oxide, β – Myrcene, Menthol, Muurolene, γ-Muurolene, trans-p-Menth-2-en-1-ol, Myrtenol, Methyl eugenol, Nerol, E-Nerolidol, (Z)-β-Ocimene, 3-Octoanone, β-Pinene, α-Phellandrene, β-Phellandrene, Palustrol, α-Selinene, β-Selinene, 7-epi-α-selinene, Sabinene, Sabinene hydrate, Spathulenol, Isosylvestrene, α-Terpinene, γ-Terpinene, β-Terpineol, α-Thujene, Thymol, Tricyclene, Verbenene and Verbenone.

Non Volatile Components:

Apart from terpenoids, the preliminary phytochemical screening showed the presence of carbohydrates, flavonoids and alkaloids in ethanolic leaf extract⁵⁹. The phytochemical analysis also showed the presence of saponins, phenols, steroids, proteins and amino acids⁴⁸. The total of seven glycosides were analytically characterized by paper chromatography and high performance liquid chromatography namely Vicenin-2 or Apigenin 6,8-di-C-glucoside, Luteolin 5-O-glucoside, Luteolin 7-O-glucoside, Quercetin 3-rutinoside, Quercetin 3-O-glucoside, Kaempferol 3-O-rutinoside and Kaempferol 3-O-glucoside were found to be present in the 80% methanolic extract The Quercetin and Kaempferol type glycosides were found to be in higher proportion which makes different flavonoid profile as compared to other species of Ocimum. The flavonoid glycosides like Luteolin 5-O-glucoside and Quercetin 3-rutinoside were also isolated from the hydroalcoholic extract which act as a marker compounds for different species of Ocimum⁶⁰. Various flavonoids like Quercetin-3,4-diglucoside and Rutin were also being reported⁶¹. The steroidal compounds present in the purified fractions of the leaves are β-sitosterol and stigmasterol⁶². It is also rich in minerals like, Sodium, Potassium, Calcium, Lithium, Iron, Copper, Manganese, Cobalt and Zinc⁴⁸. Among the different species of Ocimum, the higher amount of Ursolic acid and eugenol were also reported. The various phenolic acids like gallic acid, Protocatechuic acid, Chlorogenic acid, Caffeic acid, Ferulic acid, Sinapinic acid and Rosmarinic acid were also reported using ultra high performance liquid chromatography-hybrid linear ion trap triple quadrupole mass spectrometry⁶¹. The total phenolic component was being found to be 82.5 mg Gallic acid equivalent per gram. The various phenolic components like p-coumaric acid, Hydroxybenzoic acid, Lithospermic acid, Sinapic acid, Syringic acid and Vanilic acid were also being identified by HPLC comparison of retention time values and UV spectroscopy of the standards of the various phenolic components⁶³. The inflorescence was also found to be rich in chemical constituents like camphor, β- sitosterol, Oleanolic acid and Ursolic acid⁶⁴. The analysis of dried seeds yielded 12 to 16.5 % pale yellow fixed oil, rich in fatty acids like palmitic acid, arachidinic acid, oleic acid, linoleic acid, linolenic acid, Octa decadienoic acid and saturated acids in varying proportions. Dried seeds are also rich in crude fibre (27%), carbohydrates (23.8%), protein (18.8%) and ether extract (17.4%)^13,50. The Betulinic acid, a pentacyclic triterpenoid, anticancer agent is also reported⁶⁵.

DIFFERENT CHEMOTYPES:

O.kilimandscharicum is also known as Camphor Basil or Kapoor Tulsi due to the presence of camphor in the essential oil of its leaves. With the diversity of nature, some of the varieties replace camphor as the major ingredient of the essential oil giving rise to the different chemotypes. Camphor being the major chemotype, occurrence of 1,8-Cineole, Linalool and Eugenol chemoptype is summarized in (Table 3).

Table 3: Different Chemotypes of O. kilimandscharicum

S. No.	Chemotype	Place	Major Constituents
1.	Camphor^66,70-72	Uttarakhand, India	Camphor, Linalool, Germacrene D and Limonene
2.	1,8- Cineole⁶⁷	Rwanda	1,8-Cineole, Limonene and β-Pinene
3.	Linalool^68-71	Athens, Ohio Uttarakhand, India	Linalool, Camphor, 1,8-Cineole and (E) – caryophyllene
4.	Eugenol^70-72	Uttarakhand, India	Eugenol, 1,8-Cineole and methyl chavicol

VARIATION IN YIELD AND COMPOSITION OF PHYTOCONSTITUENTS:

The various internal factors (like gene expression, enzyme promiscuity, transcription factor, post translational modifications, presence of iso-enzyme, tissue specific regulations) different external factors (like sunlight, color shading, drying, seasonal variation, geographic location, climatic condition, rainfall, manure, fertilizer) and evolutionary and speciation factors (like habit, Anueploidy, polyploidy, chromosomal aberration, cross pollination) played a critical role in diversification of pathway which leads to the production of different metabolites^73,74. The drying of leaves increases the percentage yield of the essential oil as well as the amount of camphor^33,75. Storage of the leaves although does not affect the yield of the essential oil but may vary from place of collection. The yield and content of camphor in essential oil also varies due to season and harvesting height^38,76,77. The percentage of the volatile oil collected by steam distillation (2.36%) was higher as comparable to Hydrodistillation (1.93%) but not much difference was observed in the chemical composition⁷⁸. Enhanced exposure of ultraviolet radiations (UV-B) acted as stimulating agent for the synthesis of protein, flavonoids and hydrogen peroxide radicals, while ascorbate contents decreases at higher exposure as compared to untreated samples⁷⁹. The maximum herbage yield of camphor chemotype was observed at the initiation of seed formation (180 days after planting) but the maximum essential oil yield and content of the major constituents was being observed at the 50% seed set stage (210 days) when examined over three consecutive seasons⁸⁰. Apart from studying the effect of season, it was also reported that the volatile oil extracted after drying of the leaves showed the increase in percentage of 1,8-cineole and limonene while decrease in the percentage of camphor⁸¹. In a study conducted to determine the diurnal effect, in camphor chemotype the percentage of essential oil yield was maximum in noon (3.30%), followed by evening (3.10%) and morning (2.50%) while in the eugenol/methyl chavicol/ 1,8 cineole chemotype the oil yield decrease in the order of morning (1.36%), noon (1.25%) and evening (0.96%)⁸². Enhanced exposure of ultraviolet radiations (UV-B) acted as stimulating agent for the synthesis of protein, flavonoids and hydrogen peroxide radicals, while ascorbate contents decreases at higher exposure as compared to untreated samples⁷⁹. Principal Component Analysis (PCA) revealed the existence of a high chemical variability within the essential oils of seven species of basil. The group of components accounted for 26% of total variance, was positively correlated with 1,8-cineole, a major compound found in essential oils of O.campechianum and O. kilimandscharicum. In another group of components accounting for an additional 17% of total variance was positively correlated with camphor found in O. kilimandscharicum, and negatively with linalool⁵⁴.

TRADITIONAL OR ETHNOMEDICINAL USES:

In different parts of the world, Ocimum have been used for several purposes^83,84. Primarily, the leaves and aerial parts of the plant are used, but the combination with other plant is also being utilized by various local people of different regions. The majority of the local farmers of Kenya administered the leaves of O. kilimandscharicum along with food stuff for the protection against pests⁸⁵. The various traditional and ethnomedicinal uses across the world has been summarized in (Table 4).

Table 5: Insecticidal, Insect Repellant or Mosquito Repellant Activity

S. No.	Part Used	Organism Effected	Pharmacological Action
1.	Potted Plant⁹²	Anopheles gambiae sensu stricto Giles	Little Insect repellant
2.	Dried Leaves⁹³	Sitophilus zeamais, Rhyzopertha dominica and Sitotroga cerealella	No adult survival or progeny production,
3.	Burning of Aerial Parts^21,84	Anopheles arabiensis, Anopheles gambiae Culex quinquefasciatus, and Anopheles funestus	Deterrence effect of mosquitoes, Insect repellent
4.	Water Extract⁹⁵	Boophilus microplus and Anopheles gambiae	Acaricidal, Lowers the egg mass and hatchability percentage
5.	Silver nanoparticle (Leaf ethanol extract)⁵⁹	Aedes aegypti	Larvicidal
6.	Essential oil (Against Grain Insects) ^93,96,97	Sitophilus zeamais, Rhyzopertha dominica and Sitotroga cerealella	No adult survival, No progeny production, Toxic action, Insecticidal activity, Higher mortality rate
7.	Essential oil (Against Mosquitoes) ^21,98,99	Mosquitoes like Anopheles arabiensis, A. gambiae and Culex quinquefasciatus	Insect repellent, Biting protection, feeding inhibition and neagative oviposition activity in water
8.	Camphor (Essential oil metabolite)^55,85,100	Sitophilus granarius, Sitophilus zeamais, Tribolium castaneum, Prostephanus truncates and Helicoverpa armigera	Inhibited development of eggs and immature stages, Insect repellent, Decreased body weight and increased mortality
9.	Limonene (Essential oil metabolite)⁵⁵	Helicoverpa armigera	Decreased body weight and increased mortality
10.	β-caryophyllene (Essential oil metabolite)⁵⁵	Helicoverpa armigera	Decreased body weight and increased mortality
11.	Essential oil in glycerine and paraffin¹⁰¹	Anopheles gambiae	Feeding inhibition
12.	Decamphorized oil^7,102	Not reported	Insecticidal and Mosquito repellant

Table 6: Various Pharmacological Activities

S. No.	Pharmacological Activity	Extract / Essential Oil / Metabolite	Animal Model / Assay / Microorganisms effected
1.	Anti Diarrhoeal Activity	Aqueous Extract¹⁰³	Castor oil induced diarrheal rat model
2.	Anti Bacterial Activity	Essential Oil^53,54,71,104	Bacillus sacharolyticus, B. stearothermophilus, B. subtilis, B. thurengiensis, Enterococcus faecalis, E. faecium, Lactobacillus casei, L. plantarum, Listeria monocytogenes, L. ivanovii, Micrococcus glutamicus , Sarcina lutea, Staphylococcus aureus, S. epidermidis, Streptococcus mutans, S. pyogenes and S. viridus by disc diffusion method
		Eugenol (Essential oil metabolite) ⁶²	MDR and MRSA Staphylococcus aureus strains using disc diffusion method
		Cadinol (Essential oil metabolite)⁶²	MDR and MRSA Staphylococcus aureus strains using disc diffusion method
		Aqueous Extract²⁰	Bacillus cereus using disc diffusion method
3.	Antifungal Activity	Essential Oil^58,104,105	Alternaria sp., Aspergillus niger, A. parasiticus, Choanephora cucurbitarum, Histoplasma capsulatum, Microsporum gypseum and Rhizoctonia solani using disc diffusion method
4.	Antioxidant Activity	Methanolic extract⁶³	DPPH scavenging assay, FRAP assay, Superoxide anion scavenging activity and β-carotene-linoleic acid bleaching
		Ethyl acetate fraction (methanolic extract)¹⁰⁶	DPPH scavenging assay, FRAP assay
		Defatted hydromethanolic extract¹⁰⁷	FRAP assay
		Essential Oil⁷¹	FRAP assay
5.	Wound Healing Activity	Alcoholic extract¹⁰⁸	Incision, excision and dead space wound model in wistar rat
5.	Wound Healing Activity	Aqueous extract¹⁰⁹	Incision, excision and dead space wound model in wistar rat
6.	Anti Cancer Activity	Hydro-alcoholic extract¹¹⁰	Irradiation doses of gamma radiation in mice
		Essential Oil¹¹¹	Tumor of ovary cell lines assay
		Essential Oil⁷¹	Peritoneal macrophage cells MTT assay
7.	Immunomodulatory Activity	Methanol and Aqueous Extract¹¹²	Inhibition of HIV-1 RT and MMLV – RT
8.	Toxic and Irritant Potential Activity	Essential Oil⁵²	Immobilization of Protozoa, Application of essential oil on the skin of rabbits
9.	Antimalarial Activity	Dichloromethane extract¹¹³	SYBR Green I fluorescence assay
10.	Neurological Activity	Defatted hydromethanolic extract¹⁰⁷	MWM and EPM test in rat
11.	Polycyst Inhibition Activity	Total extract and the ethyl acetate fraction of the extract¹¹⁴	PCOS induced by letrozole in rats

SOCIO-ECONOMIC SIGNIFICANCE:

Owing to the medicinal importance Ocimum is being cultivated, utilized, evaluated and formulated in different forms throughout the world^115,116. Economy of various nations has also being affected due to the exploration as a natural source of camphor from O. kilimandscharicum. It also imparts various benefits in the field of environmental and social aspects, intellectual property rights, biotechnology aspects and other miscellaneous fields.

Natural Source of Camphor:

The therapeutic efficacy of the plant has the potential to be converted into the profitable business. During the World War II, It was being cultivated for its essential oil as a primary source of natural camphor due to which it was regarded as a substituent for Cinnamomum camphora³². 2500 lb per hectare of camphor was produced from the plant grown at an elevation of 3000 meters¹⁴. Different scientists have evolved different methods of isolation of camphor in pure form from the volatile oil of O. kilimandscharicum^69,76,115. An experimental cultivation of the plant was also being done under the Directorate of West Bengal, India for the successfully development of the method of isolation of natural camphor at a large scale. The camphor was first solidified in condenser using ice followed by the removal of oil content. The crude camphor is further purified by sublimation method. From 328 pounds of leaves, 2712 gram of pure sublimed camphor and 1136 grams of oil were obtained^118,119. In another method, the camphor was also purified by re-sublimation and the amount of camphor was determined by converting it into semi-carbazone derivative³³. The pure camphor was also collected at 63-65 ⁰C/3mm fractions when the essential oil is slowly fractionated under reduced pressure⁵⁰. Previously, it was considered uneconomical unless the oil, obtained as a byproduct also finds a commercial outlet. However, the cost of production when calculated including manpower, apparatus or machinery, cost of production, salary, depreciation charges, loss in recovery and other expenses when compared with the sale price of natural camphor favours profitable production^33,34,50. Presently, the decamphorized oil has also been utilized for its insecticidal and mosquito repellent activity⁷.

Environmental and Social Benefits:

Kakamega forest situated in the western province of Kenya was being exploited for timber, firewood, charcoal, etc. Muliru Farmers Conservation Group (MFCG) helped the farmers to cultivate the ethnomedicinal plant, O. kilimandscharicum for the production of essential oil utilized in the production of the marketed formulation. It leads to a great impact on the livelihood of the local people. Due to the domestication of plants for their medicinal properties, it generate the additional income and more than 1500 farmer makes Kshs 35,000/- to 40,000/- per year from one acre as their livelihood¹²⁰. It also has significant influence on the environment by protecting rain forest from over harvesting. It also increases the awareness of biodiversity and conservation of forests¹²¹. This helps in prevention of exploitation in Kakamega forest and improves the quality of life of the local people. The major economic impact includes the promotion of the organic plantation and starting of new micro businesses³¹. The commercial products includes Naturub Balm of 4 g, 7 g and 25 g packing used in relief from insect bite, flu, cold and chest congestion. Similarly, Naturub ointment is also available in the market in pack size of 15g and 30 g respectively useful for muscular ache and pain¹²². The inflorescence is regarded as a major source of nectar for bees in apiculture at the hills of Kilimanjaro¹⁵. The most common visitors from the bee family are Apidae followed by Megachilidae, Halictidae and Colletidae¹²³. While studying the biology and morphology of Cochlochila bullita (Stal) on O. kilimandscharicum, it was noted that eggs were laid as single/cluster over the leaf, petiole or stem. The nymph feeds on sap of leaves, inflorescence and tender stems, making the yellow spot initially which turns into brown color confirming its another importance in an ecological system¹²⁴. Without the variation in essential oil component, the vigorous growth of plants without exhibiting any morpho-phytoxicity is observed when grown in metal rich soil like copper, cadmium and lead raising the opportunity to grow in area contaminated with metal¹²⁵.

Intellectual Property Rights:

The different species of Ocimum including O. kilimandscharicum is being patented for its usage to control obesity. The basis for the patent is the study of the fruit O. canum which is neutral and good dieting agent. It does not cause feeling of hunger, neither decreases the appetite. It also does not show any adverse reaction when taken for long period and could also be preserved as a dry fruit¹²⁶. The essential oil is also being patented as disinfectant against many pathogenic micro-organisms for potable water¹²⁷. Another patent claims that the product prepared using combination of Cinnamon and Ocimum extract of any species or variety to help to treat, prevent or improve the urinary infection¹²⁸. The agriculture composition including the essential oil of O. kilimandscharicum claims the active ingredients present in the essential oil enhances the metabolic activity and the protection of the plant against microbes or pests¹²⁹. As the Eugenol and β-Caryophyllene is present in the essential oil of various plants including O. kilimandscharicum. The formulation containing eugenol and β-caryophyllene and/or their derivatives and/or analogues have been patented for the management of pain and inflammation¹³⁰. The US patent was being granted for the development of the method used in detecting mercury ion in water solution. The various species of Ocimum including O. kilimandscharicum was claimed for the preparation of the reagent which includes silver nanoparticles and Ocimum extract. The method was based on the principle of change in color after the addition of reagent in water¹³¹. Another US patent was being granted based on similar principle but being utilized for the detection of mercury in water based compositions¹³². The essential oil comprises numerous components which are toxic for the pests or fungus affecting the bees. After confirming its non toxic effect on the bees, the essential oil or the preparation comprising essential oil is patented for the management of bee disease or pests¹³³. The oral care formulation is being patented comprising the extracts of various herbs including O. kilimandscharicum to be used as herbal dentifrice¹³⁴. The application has been drafted for the formulation ‘Pancha Tulasi Drops’, comprising of combination of essentials oils of Ocimum species including O. kilimandscharicum with the claim of disinfecting potable drinking water¹³⁵. Another herbal preparation is claimed for patent comprising Rhododendron arboretum, O. kilimandscharicum and Citrus limon for the prevention and treatment of depression with lesser side effects at minimal cost¹³⁶.

Biotechnological Aspects:

The essential oil of fresh leaves collected after inoculated with biofertilizers like Glomus fasciculatum, Azotobacter chroococcum and Aspergillus awamori revealed the increase in percentage yield of oil from 0.6 % to 2.03 % v/w. The components like α- pinene and linalool were identified after inoculation along with increase in percentage of camphene, camphor and eugenol¹³⁷.The combination of 2,4-dichlorophenoxyacetic acid, α-naphthaleneacetic acid (NAA) and kinetin favours the callus growth with the production of 1.87% Betulinic acid, a pentacyclic triterpenoid which is proven anticancer agent without damaging the normal body cells. The use of methyl jasmonate has also increased the percentage yield of betulinic acid⁶⁵. Nodal segment was excised and plantlet formation was being observed of the fertile hybrid of O. basillicum X O. kilimandscharicum by tissue culture method using Murashige and Skoog’s (MS) revised tobacco medium in combination with different plant growth regulators. Shoots were developed using 5 X 10^-6M Naphthaleneacetic acid (NAA) while Benzyl adenine (BA) of 5 X 10^-6M developed 2-10 roots. The combination of Benzyl Adenine (5 X 10^-6M) and NAA (10^-5M) development of 30-40 differentiated roots while the combination of BA (5 X 10^-6M) and Indole acetic acid (IAA) (10^-5M) shows 20-30 differentiated roots after 5 weeks. The callus formation takes place with the combination of Benzyl adenine (5 X 10^-6M) and IAA (5 X 10^-6M or 10^-6M)¹³⁸. MS medium mixed with 1.0 mg/l of 6-BA showed the maximum regeneration (93.88%) and maximum number of multiple shoots (6.09±0.05), with average length 3.83±0.11cm in auxiliary shoot bud proliferation. Half-strength MS medium supplemented with 1.5mg/l Indole-3-butyric acid and with reduced sucrose concentration (2%) gave us well developed roots within 20–25 days. Random amplified polymorphic DNA (RAPD) markers also established the clonal fidelity and genetic stability of mother plants and the plantlets regenerated from nodal stem segments confirming the method to be used for large scale propagation¹³⁹. The healthy propagation of stem were obtained by 20cm long cutting of the medium portion. For the better growth 2000ppm of IAA is the most suitable auxin. The use of combination of Inorganic fertilizers like Di-ammonium phosphate (2.1g), Urea (2.1g), Muriate of Potash (1.9g) per plant yields maximum growth and yield of the essential oil. The usage of Biofertilizer in combination of Azotobacter (3 g), Phosphate solubilizing bacteria (3g) and Farm Yard Manure (60g) also gave the high herbage yield along with maximum growth of plant¹⁴⁰. The best composition for the encapsulation of shoot tip was possible using sodium alginate (3%) and calcium chloride (75mM) producing clear and iso-diametric artificial seeds. Maximum regeneration of shoots was possible after 6 weeks in 0.7% solidified agar, MS medium and 1mg/l BA (butyric acid) followed by changing the composition of the medium to half MS Medium and 1.5mg/l BA for 4 weeks required for root formation. After the formation of both root and shoot, the explants were transferred into equal mixture of soil and vermiculite. After 2-3 weeks transplantation was done in larger pots comprises of soil and organic manure with the survival rate of 79.53%. Genetic stability was confirmed using PCR based molecular markers like RAPD (random amplified polymorphic DNA) and ISSR (Inter specific sequence repeat) amplification profile which shows no difference in banding pattern taken from mother plant, stored seeds and regenerated plants¹⁴¹. The presence of glandular as well as non glandular trichomes and the increase in the proline content of the hybrid developed from the basilicum and kilimandscharicum species of the Ocimum, make it possible to survive against cold. Increase in gene expression WRKY and MPS (microspore-specific), has also been reported which provides response against cold¹⁴². The over expression of 3-hydroxy-3-methylglutaryl Co-A reductase (HMG-CoA) isolated from the O. kilimandscharicum increased the production of terpenoidal constituents of essential oil in other species like basilicum, sanctum and gratissimum. It also increases the carotenoid content of Artemisia annua, and Withania somnifera. The increased content of artemisin in A. annua and total withanolide in W. somnifera was also observed¹⁴³.

Miscellaneous Benefits:

Enormous experimentation had shown the valuable benefits that could be used for the commercial exploration of other valuable marketed products. The solutions of the camphor rich essential oil obtained from O. kilimandscharicum and Cinnamomum camphora solublises various insecticides like like DDT and Benzene hexachloride with increased toxicity effect against larvae of Culex fatigans which shows that these types of oil can be used as solvent in mosquito control operations¹⁴⁴. Four years old essential oil is competitively similar to the synthetic repellent N, N-diethyl-3-methylbenzamide (DEET) conforming that the storage do not decreases the potency, hence could be used effectively for making marketed formulations¹⁰¹. The usage of the seed oil in paint is superior to linseed oil as it shows the rapid drying, harder film along with bright finish⁵⁰. Low boiling fraction of the oil may be used as a solvent and vehicle for metallic luster on ceramic bodies¹⁰². It is one third as effective as pyrethrum extract¹³. The formulation of herbal syrup using the decoction of the leaf also confirmed the antimicrobial and antioxidant activity¹⁴⁵. However, camphor which is the major ingredient of the essential oil of O. Kilimandscharicum is utilized for various disorders as per the ayurvedic literature like flatulence with gurgling sound, digestive impairment, rheumatism, tastelessness, diarrhoea, burning sensation, pyorrhoea, toothache, chronic sinusitis, itching, disease of throat, male impotence, helminthiasis/worm infestation, diseases of skin, obesity, intercostal neuralgia and pleurodynia, joint pain, asthma, thirst, skin diseases, eczema, disorders due to poison, gastro-enteritis with piercing pain, renal disorder¹⁴⁶.

CONCLUSION:

The present manuscript describing the plant attribute will help the researchers, reviewers, scholars and the common man to understand the concepts involved in the study of medicinal plant. The ethnomedicinal use along with proven facts had helped in development of exclusive products. Further investigations are required for the isolation of the phytoconstituents responsible for pharmacological activities which may help in the development of novel molecules for the treatment of diseases. Apart from medicine, the current knowledge in vast domain has also asserted its potential and utility for commercial purposes in different industries will surely amplify the economy.

ACKNOWLEDGEMENT:

We wish to thank IKG Punjab Technical University, Kapurthala for providing the opportunity for the upliftment of educational qualification and also granting access an OpenAthens facility which enables to raise the standard of education.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 21.12.2019 Modified on 05.02.2020

Research J. Pharm. and Tech. 2020; 13(10):4751-4764.

DOI: 10.5958/0974-360X.2020.00837.9

S. No.	Traditional Use	Part Used or Combination with Other Plant	Region or Country
1.	Anorexia¹²	Leaves	India
2.	Aromatic, Flavoring Agent^15,36	Leaves	India
3.	Bacterial and Viral infections¹²	Leaves	India
4.	Bronchial asthma^10,15,36	O.kilimandscharicum and Black Pepper	India
5.	Catarrh, Cough and Cold^12,18,86	Leaves or Vapor of boiling/fresh leaves	India, Kenya, Tanzania
6.	Congested chest, Bronchitis^10,18,86	Leaf or Vapor of boiling/fresh leaves or leaf with or without black pepper	Kenya, Brazil
7.	Eye Infections⁶⁷	Leaves	Rawanda
8.	Fever^15,36	In combination with Black Pepper	India
9.	Flu^10,18,36	Aerial Parts, In combination with Lippia ukamens or black pepper	Brazil, Kenya, India
10.	Foul body smell³⁶	boiled water of leaves	India
11.	Halitosis or Bad breadth¹²	Leaves	India
12.	Headache⁴¹	Leaf	India
13.	Insect repellents for Malaria menace^17,18,21,36	Aerial Parts, Fresh / Smoke of leaves	Kenya, Tanzania, India
14.	Measles¹⁸	Infusion of leaves	Africa
15.	Protection against pests in food stuff^15,35,85	Aerial Parts/ Leaves	Kenya, Tanzania, India
16.	Sinus Problems⁴¹	Leaf	India
17.	Skin diseases like ringworm and scabies³⁶	Boiled water of leaves	India
18.	Stomachache or distention of abdomen^12,18	Leaves or in combination with Lippia ukamens	India, Kenya
19.	Ulcers^12,36	Leaves or boiled water of leaves	India
20.	Wound Healing^10,12,36	Aerial Parts, Leaves or boiled water of leaves	Brazil, India