Bioactive Compounds via in vitro Culture Approach and Pharmacological Attributes of Genus Euphorbia : A Comprehensive Review

: The family Euphorbiaceae comprises 2000 species and is listed as third among the largest flowering family. Euphorbia is used in traditional treatment for various diseases, including dengue fever, dysentery, diarrhea


INTRODUCTION
Family Euphorbiaceae contains herbs, shrubs, trees, and succulent plants; both wild and cultivated plants of this family are found all over the globe [1].Most of the species of this family chiefly existed in tropical regions but also predominantly extended into temperate zones.Most species of the family Euphorbiaceae produce milky latex; reported as poisonous in several species (Euphorbia tirucalli, Euphorbia royleana, Euphorbia lathyris, Euphorbia esula, Euphorbia conitofolia Euphorbia milii, Euphorbia hirta, Euphorbia neriifolia, and Euphorbia heliscopia) as described previously [2][3][4][5][6].In Pakistan, the family Euphorbiaceae is comprised of 24 genera and 90 species.Besides this, extracts obtained from various parts of the genus Euphorbia proved their efficiency in relieving constipation and other gastrointestinal disorders.Moreover, its cytotoxic property bears antineoplastic compounds in the forthcoming time [7].The traditional uses in medicine and industry of the latex obtained from Euphorbia species are well-known.Different members of this family are extensively used for aesthetic and medicinal purposes and cultivation has significant economic importance [8].
Euphorbia hirta has been investigated with diverse medicinal properties; conventionally, used for curing different ailments like dengue fever, diarrhea, dysentery, ulcer, asthma, bronchitis, etc.The latex obtained from E. hirta is extensively used in treating jaundice, anemia, and skin disorders.Similar antipyretic potential appeared in Euphorbia neriifolia [9].Moreover, Awaad et al. [8] investigated several pharmacological activities from different parts of E. hirta extracts, which include antispasmodic, antifungal, antibacterial, anticatarrhal, diuretic, etc. Folk medicine in Australia utilized the latex obtained from Euphorbia peplus for curing keratosis and skin cancer [10,11].Recently, it is widely used as an essential part of many natural and medicinal products [12].A huge number of pharmacological attributes have also been reported from Euphorbia tirucalli [13].
Tissue culture could be defined as the sterilized culture of cells, tissues, and organs in a controlled condition.Plant tissue culture is also known as sterile culture, in vitro culture, or axenic culture; an essential and fundamental technique in commercial and applied studies [14,15].Plant tissue culture media contain vitamins, micronutrients, macronutrients, and all other essential components required for normal growth and development.The pH recommended for the proper culturing of cells and tissues ranges between 5.3 and 5.8 [16].Auxin's badly affecting the in-vitro morphogenesis of Euphorbia nivulia.Similarly, Martin et al. [17], studied the effect of auxins and cytokinin on hypocotyltyle culture of Euphorbia esula.Plant tissue culture is becoming a pressing need of the hour to conserve endangered species through clonal propagation and production of medicinally important plants on large scale in a controlled condition in a well-defined aseptic way [18,19].Extensive research has been carried out and still improvements need in the in-vitro culture technique in boosting the yield of secondary metabolites.In this literature review, we have discussed the efforts made by different phytochemists, botanists, pharmacologists, biochemical engineers, and tissue culturists for the improvement and establishment of the in-vitro cultivation approach of Euphorbia species.In a parallel review documented only the biological activity and triterpenoids content of the genus Euphorbia; however, no review analyzed the procedures for the preparation of different explant cultures, in-vitro production of biologically active compounds from various cultures, and enlisting the detailed pharmacological attributes of genus Euphorbia [20].

DATA COLLECTION STRATEGY
The information regarding this review was systematically collected from different scientific databases including Google Scholar, Science Direct, PubMed, and published literature.The papers selected from the base were most suitable on keywords: biological activities, pharmacological effects, Euphorbia, phytochemicals, and disease treated without time limitation.The 149 publications that were chosen, spanning the years 1978 to 2022, displayed remarkable findings that reflected current scientific trends at the time of publication.

PHYTOCHEMISTRY AND PHARMACOLOGY OF GENUS EUPHORBIA
Conventionally, the genus Euphorbia is used in treating some ailments, owing to its astonishing disease-curing properties.
Recently the mesmerizing therapeutic potential of the genus Euphorbia has startled researchers, several pure compounds were also isolated from extracts of different plant parts [21].The phytochemical screening of genus Euphorbia showed the presence of essential phytochemicals having robust therapeutic influence, other than its conventional uses it has robust medicinal properties [22], such as anti-arthritis, anti-diarrheal, analgesic, hepatoprotective and antipyretics (Table 1 in Annexure I).We provided an overview of various parts of the species belonging to the genus Euphorbia (Figures 1 and 2), reflecting the importance of various parts in context to their pharmacological potential.
Whereas, the applications of whole plants or various plant parts on different animals'/cell lines have been documented (Figure 3).In addition, flavonoids, diterpenoids, triterpenoids, tannins, and polyphenols were also being isolated from some species of Euphorbia through phytochemical screening.As far as their biological activities are concerned; phytochemical responses vary greatly, in most cases diterpenoids showed anti-cancer and cytotoxic activity; flavonoids and triterpenoids proved effective in treating inflammation and inhibiting pathogenic activities.Many natural products have been derived from Euphorbia; mainly including essential oils, pure compounds, and extracts with promising biological activities.About 80 phytochemicals have been reported from essential oils of Euphorbia species and prominent secondary metabolites [23].Furthermore, E. hirta leaves extract confirmed the presence of essential vitamins in sufficient amounts, including vitamin B2, vitamin E, and vitamin C [24].

IN VITRO TISSUE CULTURE APPROACHES IN GENUS EUPHORBIA
Family Euphorbiaceae contains a large number of species; including many endangered and endemic species.Though, in vitro cultivation is limited to some specific genera having medicinal, aesthetic, food, rubber, and dye-yielding purposes [25].In vitro culture is a conducive and effective technique for the proliferation and conservation of endangered species in a shorter period, mainly for those plants which are difficult to be grown by using conventional methods of cultivation and conservation [26].Plants regenerated through in vitro culture possess some advantageous features over those cultivated in fields via conventional agricultural practices.For instance, the culturedgrown mountain arnica rhizome has a characteristic smell and taste, lacking in the same plant rhizome cultivated in field conditions.Several active phytochemicals were isolated from different plant parts and propagated via cell, tissue, and hair root culture methods [27].It is immensely important to select a parent plant with a considerable amount of biologically active phytochemicals for callus formation; likewise, selecting those cell lines with higher yield [28]. Succeeding reports on cell culture, callus culture, shoot, leaf, and root culture, somatic embryogenesis, and nodal/inter nodal culture of genus Euphorbia (Table 2 in Annexure II).

CALLUS CULTURE OF GENUS EUPHORBIA
A callus is a mass of tissues having differentiated cells, developed under the influence of determinate hormonal control described previously [29,30].Propagated callus and shoot from stem pieces of Euphorbia esula HR lines and shoot regeneration from hypocotyls of non-HR lines.Maximum shoot regeneration was observed by inoculating the explants in a growth medium containing Murashige and Skoog (MS) basal salts, MS + vitamins, 1.11 μM 6-benzylaminopurine, 1.97 μM indole-3butyric acid, and 3.0 % sucrose, pH 5.6-5.8.After 30 days multiple shoots developed from the stem (Figure 4).

In vitro Culture Approach and Pharmacological Attributes of Genus Euphorbia
of the genus Euphorbia has startled researchers, several pure compounds were also isolated from extracts of different plant parts [21].The phytochemical screening of genus Euphorbia showed the presence of essential phytochemicals having robust therapeutic influence, other than its conventional uses it has robust medicinal properties [22], such as anti-arthritis, anti-diarrheal, analgesic, hepato-protective and antipyretics (Table 1 in Annexure I).We provided an overview of various parts of the species belonging to the genus Euphorbia (Figures 1  and 2), reflecting the importance of various parts in context to their pharmacological potential.
Whereas, the application of whole plants or various plant parts on different animals'/cell lines has been documented (Figure 3 in Annexure II).In addition, flavonoids, diterpenoids, triterpenoids, tannins, and polyphenols were also being isolated from some species of Euphorbia through phytochemical screening.As far as their biological activities are concerned; phytochemical responses vary greatly, in most cases diterpenoids showed anti-cancer and cytotoxic activity; flavonoids and triterpenoids proved effective in treating inflammation and inhibiting pathogenic activities.Many natural products have been derived from Euphorbia; mainly including essential oils, pure compounds, and extracts with promising biological activities.About 80 phytochemicals have been reported from essential oils of Euphorbia species and prominent secondary metabolites [23].Furthermore, E. hirta leaves extract confirmed the presence of essential vitamins in sufficient amounts, including vitamin B2, vitamin E, and vitamin C [24].

In vitro Culture Approach and Pharmacological Attributes of Genus Euphorbia
The callus culture of Euphorbia hirta revealed the presence of phenolic and sterol compounds with a substantial amount of chlorogenic acid (79.67 mg/100 g d. m.) syringic acid (32.57mg/100 g d. m.) and brassicasterol (32.57mg/100 g d. m.) by Özbilgin et al. [31] and Lone et al. [32].Euphorbia tirucalli callus culture revealed the presence of euphol, tirucallol, and 4, 4-dimethyl sterols amount [33,34].The most prominent secondary metabolites extracted from cultured cells of Euphorbia species include cyanidin glycoside from Euphorbia milli.Similarly, the sitosterol, palmitic acid, and triterpenoids from cultured cells of E. esula, and phytosterol, tirucallol, triterpene, and euphol (Table 3 in Annexure III) extracted from callus cultured cells of Euphorbia tirucalli [33,35].Euphorbia characias callus culture hormonal regulation of triterpinols formation was investigated [36].Leaf explants of E. hirta were cultured on MS+NAA and 6-benzylaminopurine (BAP) medium, at the onset of callus initiation, it was again subcultured on the same media with 1   mg/L concentration of NAA (1-naphthaleneacetic acid) and BAP [30].Red callus was produced from apical and axillary buds of E. pulcherrima on MS basal medium containing benzyladenine (BA) and a combination of IAA (indole acetic acid) (IAA) and BA [37].From leaf explant of E. helioscopia callus was induced via Murashige and Skoog's (MS) medium supplemented with 6-benzylaminopurine [38].Furthermore, secondary metabolite extraction is of utmost importance in culturally grown plants; making it an ideal technique for raising plants for commercial and medicinal purposes [29].

SHOOT, LEAF, SEED, AND ROOT REGENERATION VIA DIFFERENT CULTURES
Murashige and Skoog's (MS) + naphthaleneacetic acid (NAA) medium was used to produce roots from E. tannensis shoot culture, some of the seedlings were kept under glasshouse in pots containing coarse sand, peat, and perlite with a ratio of 6:3:1, respectively.The plantlets remained healthy and grew well under the glasshouse.On contrary, when placed in peat blocks all seedlings wilted and collapsed.In comparison with E. tannensis; in-vitro root formation from shoots of E. lathyris was quite slow on the same medium used for the formation of roots from E. tannensis shoots [39].Euphorbia antisyphilitica shoots were propagated via invitro culture technique by using BAP (4.44 μM) and MS + NAA (0.13 μM) medium, root progress was satisfactory and when transferred to the field; easily adapted to the natural climatic conditions [31].Euphorbia lagascae shoots were regenerated through tissue culture technique; afterward, dipped in IBA (50 mg/L) for a period of 2 minutes, an increase of 70% to 100% survival rate was observed with the application of benzyladenine (BA) [40].Tips of Euphorbia pugniformis cristate lateral shoots were cultured by using MS + NAA (0.1 mg/L) sucrose 2.0% and IBA in culture media; as a result, both of the normal and cristate types of shoots were produced.90%-100% of cultured plantlets successfully acclimatized outside the laboratory in field conditions [41].In-vitro propagation of Euphorbia fulgens micro shoot cuttings and their adjustment to the natural climatic conditions were established [42].Euphorbia pulcherrima shoot buds were propagated through the tissue culture technique [43].Shoots were raised from nodal shoot explant of Euphorbia pulcherrima (Figure 5) using a medium having 6-benzylaminopurine (BAP) in combination with adenine sulfate and GA3 (Gibberellic acid).
The induction of shoot was optimal by using BAP at 0.5 mg/L in combination with 20 mg/L adenine sulfate.Numerous roots and the highest frequency (77.8%) were observed by using MS media and supplements of 1.0 mg/L indoleacetic acid (IAA) by Sreenika et al. [44].E. esula hypocotyl segment was applied as explant and roots were proliferated on culture media containing IAA and IBA [45].Leaf-cultured cells of Euphorbia milli showed the presence of a red colour pigment mainly consisting of anthocyanin [46,47].Extract of leaves of Euphorbia cotinofolia in streptomycin possessed wide range of flavonoids, terpenoids, and steroids that help remove a pathogenic form of Bacillus cereus, Bacillus subtilis, Escherichia coli, e cultivated in field conditions.Several active hemicals were isolated from different plant parts opagated via cell, tissue, and hair root culture s [27].It is immensely important to select a parent 3.0 % sucrose, pH 5.6-5.8.After 30 days multiple s developed from the stem (Figure 4).Enterobacter aerogenes, Klebsiella pneumoniae, Salmonella typhi, and Staphylococcus aureus [48].Amorphous calcium phosphate nanoparticles (ACP NPs) with coumarin extracts from seeds of Euphorbia lathyris possessed strong cytotoxicity against colon cancer [49].

SOMATIC EMBRYOGENESIS NODAL AND INTER NODAL CULTURES OF GENUS EUPHORBIA
Cultivation through somatic embryogenesis provides a prospect to propagate those lines bearing superior quality in terms of secondary metabolites and yield attributes [43].To date, propagation through cell suspension culture, callus culture, and somatic embryogenesis was described in several Euphorbia species.The somatic embryogenesis of Euphorbia pulcherrima was investigated [42].
Euphorbia pulcherrima nodal explants were cultured on NAA + MS, isopentenyl adenine (2-iP), and Kin media [52].Euphorbia pulcherrima nodal explant on MS + α-naphthalene acetic acid and isopentenyl adenine (2-iP) medium gave rise to somatic embryos [53].Internode explant of E. hirta gave rise to a higher number of somatic embryos on BAP and Kin media supplemented with indole-acetic acid (IAA) and naphthalene acetic acid (NAA).NAA proved more productive than indole-acetic acid (IAA) with a higher percentage of somatic embryos.100% response was noticed by using MS in combination with 0.5 mg/L naphthalene acetic acid (NAA), 0.4 mg/L each of BAP, and kinetin (Kin).Medium with IAA in place of NAA gave maximum (92%) somatic embryo induction [54,55].

INFLORESCENCE TISSUE CULTURE
Das et al. [56] manipulated the inflorescence of explants (Euphorbia milli) for their in-vitro propagation.Consequently, vegetative meristems were cultivated from the meristems of an inflorescence of the main axis after one week of inoculation on MS medium supplemented with indole-3-butyric acid (IBA) and benzyl adenine (BA).Among various growth regulators, MS medium with 1.0 mg/L BA and 0.3 mg/L IBA responded better in terms of callus initiation with a maximum percentage of leaf and shoot developed.

CONCLUSIONS
In this review, pharmacological activities, methodologies of preparing different explant cultures, in-vitro production of biologically active compounds, and vital phytochemicals extracted from various parts of the genus Euphorbia has been documented.For successful in-vitro propagation of seedlings; media composition, plant growth regulators (PGRs) selection, and other vital requirements such as temperature, light, and pH are immensely important to be determined.Moreover,  The induction of shoot was optimal by using BAP at 0.5 mg/L in combination with 20 mg/L adenine sulfate.Numerous roots and the highest frequency (77.8%) were observed by using MS media and supplements of 1.0 mg/L indole-acetic acid (IAA) by Sreenika et al. [44].E. esula hypocotyl segment was applied as explant and roots were proliferated on culture media containing IAA and IBA [45].Leaf-cultured cells of Euphorbia milli showed the presence of a red colour pigment mainly consisting of anthocyanin [46,47].Extract of leaves of Euphorbia cotinofolia in streptomycin possessed wide range of flavonoids, terpenoids, and steroids that help remove a pathogenic form of Bacillus cereus, Bacillus subtilis, Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Salmonella typhi, and Staphylococcus aureus [48].Amorphous calcium phosphate nanoparticles (ACP NPs) with coumarin extracts from seeds of Euphorbia lathyris possessed strong cytotoxicity against colon cancer [49].

SOMATIC EMBRYOGENESIS NODAL AND INTER NODAL CULTURES OF GENUS EUPHORBIA
Cultivation through somatic embryogenesis provides a prospect to propagate those lines bearing superior quality in terms of secondary metabolites and yield attributes [43].
To date, propagation through cell suspension culture, callus culture, and somatic embryogenesis was described in several Euphorbia species.The somatic embryogenesis of Euphorbia pulcherrima was investigated [42].From the hypocotyl of Euphorbia pulcherrima somatic embryo formation was reported by Biesboer et al. [50].Euphorbia tirucalli internodal explants (Figure 6) were propagated via in-vitro culture technique by using Linsmaier and Skoog's (LS) + TDZ (0.02 mg/L) culture medium [51].
Euphorbia pulcherrima nodal explants were cultured on NAA + MS, isopentenyl adenine (2-iP), and Kin media [52].Euphorbia pulcherrima nodal explant on MS + αnaphthalene acetic acid and isopentenyl adenine (2-iP) medium gave rise to somatic embryos [53].Internode explant of E. hirta gave rise to a higher number of somatic embryos on BAP and Kin media supplemented with indole-acetic acid (IAA) and naphthalene acetic acid (NAA).NAA proved more productive than indole-acetic the plants regenerated through tissue culture need to be assessed for their safety in living organisms is also necessary for higher marketability and significance.Besides this, investigating the potential of preventing diseases by applying Euphorbia should be a relevant issue.Disease prevention methods and modern cultivation practices for Euphorbia need to be further elucidated with the application of advanced techniques like hydroponic, aeroponic, and plugged seedling culture methods that should be adopted for rapid growth and better quality of Euphorbia species.Still, there is a great need for an imperative propagative method and an immense desire for the swift proliferation of superior qualities of Euphorbia species.

FUTURE PERSPECTIVES AND RECOMMENDATIONS
The identification and isolation of novel bioactive chemicals from the genus Euphorbia should be the focus of future study.Analytical advances such as mass spectrometry and nuclear magnetic resonance can help with the isolation and structural elucidation of previously unknown substances.Further optimisation of in vitro culture conditions is required to maximise bioactive chemical synthesis.This includes optimising nutrient formulas, growth regulators, and culture medium components to mimic the natural environment and increase target compound yield.Exploring biotechnological technologies for large-scale synthesis of bioactive substances, such as plant cell and tissue culture, can provide a sustainable and controlled supply.This could include the development of bioreactor systems and commercial scale-up initiatives.More thorough pharmacological investigations on isolated chemicals from Euphorbia species are required to fully grasp their medicinal potential.Following clinical trials, their efficacy, safety, and prospective applicability in treating various diseases can be validated.
Promote interdisciplinary collaborations between geneticists, pharmacologists, biotechnologists, and other experts.Such collaborations have the potential to speed research and bring varied perspectives to bear on the issues connected with the study of Euphorbia species as well as establish standardized protocols for the extraction of bioactive compounds from Euphorbia plants.This will assure uniformity in research outputs and make it easier to compare results across investigations.Finally, the possibilities for future study on bioactive substances from the genus Euphorbia are encouraging, with opportunities for scientific improvements, biotechnological innovations, and sustainable practices.Implementing the recommended procedures can aid in the discovery of novel treatments, the conservation of plant species, and the promotion of responsible resource use.

CONFLICT OF INTEREST
The authors declare no conflict of interest.acid (IAA) with a higher percentage of somatic embryos.100% response was noticed by using MS in combination with 0.5 mg/L naphthalene acetic acid (NAA), 0.4 mg/L each of BAP, and kinetin (Kin).Medium with IAA in place of NAA gave maximum (92%) somatic embryo induction [54,55].

INFLORESCENCE TISSUE CULTURE
Das et al. [56] manipulated the inflorescence of explants (Euphorbia milli) for their in-vitro propagation.Consequently, vegetative meristems were cultivated from the meristems of an inflorescence of the main axis after one week of inoculation on MS medium supplemented with indole-3-butyric acid (IBA) and benzyl adenine (BA).Among various growth regulators, MS medium with 1.0 mg/L BA and 0.3 mg/L IBA responded better in terms of callus initiation with a maximum percentage of leaf and shoot developed.

CONCLUSIONS
In this review, pharmacological activities, methodologies of preparing different explant cultures, in-vitro production of biologically active compounds, and vital phytochemicals extracted from various parts of the genus Euphorbia has been documented.For successful in-vitro propagation of seedlings; media composition, plant growth regulators (PGRs) selection, and other vital requirements such as temperature, light, and pH are immensely important to be determined.Moreover, the plants regenerated through tissue culture need to be assessed for their safety in living organisms is also necessary for higher marketability and significance.Besides this, investigating the potential of preventing diseases by applying Euphorbia should be a relevant issue.Disease prevention methods and modern cultivation practices for Euphorbia need to be further elucidated with the application of advanced techniques like hydroponic, aeroponic, and plugged seedling culture methods that should be adopted for rapid growth and better quality of Euphorbia species.Still, there is a great need for an imperative propagative method and an immense desire for

Fig. 4 .
Fig. 4. In vitro regeneration of leafy spurge (Euphorbia esula L.) a = Large callus with a loose structure, b = Shoot regeneration from hypocotyls of non-HR lines, c = Shoots regeneration from stem pieces of HR lines, d = Callus, e = Callus and shoot primordium, f = Multiple shoots regenerated from the wounded surface of explants, g = Multiple plantlets growing from calluses, h = Plantlets developing roots in rooting medium.(i) Plantlets growing In-vitro.
In vitro regeneration of leafy spurge (Euphorbia esula L.) a = Large callus with a loose structure, b = ration from hypocotyls of non-HR lines, c = Shoots regeneration from stem pieces of HR lines, d = Callu and shoot primordium, f = Multiple shoots regenerated from the wounded surface of explants, g = Mu ts growing from calluses, h = Plantlets developing roots in rooting medium.(i) Plantlets growing In-vitro In vitro Culture Approach and Pharmacological Attributes of Genus Euphorbia 571

antiquorum f In vitro Culture Approach and Pharmacological Attributes of Genus Euphorbia Fig. 3. Percentage
of animals/cell lines used for the assessment of various pharmacological activities of euphorbia genus different parts extracts (a) Euphorbia hirta (b) Euphorbia nerrifolia (c) Euphorbia kansui (d) Euphorbia helioscopia (e) Euphorbia tirucalli (f) Euphorbia antiquorum (g) Euphorbia milli (h) Euphorbia pulcherrima (i) Euphorbia characias.

Table 3 .
In-vitro production of secondary metabolites from Euphorbia species via using different approaches.