In vitro Establishment of Saussurea costus – An Endangered Medicinal Herb

The taxonomic classification of S. costus is as follows.

S. costus is a lean, evergreen plant and can be as long as 2 m. The feature of the plant also includes the presence of certain fibers whereas the root of the plant can grow up to 60 cm long and emits a unique aroma; It possesses concave, bent, flat, and hairy fruits; organized in terminal and apical clusters (M. M. Pandeyet al., 2007). Plant flowers are navy in color have membranes, are clustered, and possess uneven bumps; upper leaves are small while leaves at the base are larger and have lobes along with a stretched-out winged stack (Zaharaet al., 2014).

Around 62 species of S. costus are indigenous to the Indian Himalayan Region and hold significant medical importance (Rathoreet al., 2021). Saussurea costus (synonym Saussurea costus lipsch) is found at an altitude of 3000-3500 m higher than the sea level in the region of the Northwestern Himalayas. It is also finely distributed in certain states of India such as Kashmir, Uttarakhand, etc., (Gwariet al., 2013) as well as in certain regions such as Suru Valley in Ladakh, Kishenganga, and the higher altitude regions such as the Chenab valleys in Jammu and Kashmir (Kalaet al., 2006).

Phytochemicals are the primary or secondary metabolites that the plant produces, they are not just important for the better development of the plant but also protect it from harmful agents (Martinezet al., 2017). Phytochemicals are proven to help in treating various human illnesses. Phytochemicals are reported to demonstrate chemoprevention and chemotherapeutic activities in cancer cell lines and animal models (Bathaieet al., 2015).

The roots of S. costus contain various phytochemicals such as monoterpenoids, dehydrocostus lactone, flavonoids, glycosides, triterpenes, etc. (Rathoreet al., 2021). It also contains alkaloids, anthraquinones, costunolide, dihydrocostus lactone, etc. (Zaharaet al., 2014). The roots of the S. costus are particularly abundant in the sesquiterpenoid (Rathoreet al., 2021). Sesquiterpenoids are present in a larger proportion (79.80%) of the essential oil derived from roots than monoterpenoids (13.25%), Dehydrocostus lactone is also a significant component of the S. costus (Liuet al., 2012).

Dehydrocostus lactones and costunolide have shown prevention against breast cancer (Penget al., 2017) and they are also proven to be effective against a variety of microbiological diseases. Dehydrocostus lactone can treat the disintegration of cells, growth restriction, and antibacterial attributes (Pandeyet al., 2007);(Penget al., 2017). Dehydrocostuslactone has an impact on apoptosis and its mechanism of action in human leukemia cells (Chang and Kim, 2008). Potential anti-cancer drugs contain various phytochemicals such as costunolide, and dehydrocostus lactone as their constituent (Chenet al., 2008; Choet al., 2004; Jeonget al., 2002). Several reports point out the idea that the herb-S. costus has anti-parasitic, anti-diabetic, CNS depressive, and hypolipidemic qualities (Rathoreet al., 2021).

The shoot system of the S. costus acts as both lubricant and feed in the district of Himachal Pradesh (Butola and Samant, 2010). Its roots are used as perfume and as a pesticide in the Indian Himalayan region to protect wool clothing from insects (Rathoreet al., 2021), along with that it is also been used in antiseptic, hair oils, high-quality perfume formulations and locally it used for curing stomachaches, colds, tooth pain, ulcers, etc. (Rathoreet al., 2021). S. costus has been recommended for noncyclic menstruation and lower body pain (S. G. Koet al., 2005).

The extracts from the S. costus, such as costunolide have the potential to develop into potent drugs for cancer treatment as there are numerous of evidence that points out their anticancerous effect (Rasulet al., 2012). Dihydrocostus lactone present in its roots has shown a significant effect in the suppression of the development of cancer cells (Linet al., 2015). The phytochemicals extracted from the S. costus leaves showed impressive anticancerous effects on the cancer cell lines, these phytochemicals arrest the cell cycle in the G1 phase, halting the DNA replication, and inducing apoptosis (Rathoreet al., 2021). S. costus extracts activate the mitochondrial pathway which increases the level of pro-apoptotic proteins decreases the level of antiapoptotic protein expression level and induces apoptosis (Shatiet al., 2020).

Figure 1:
Flowchart representing different techniques in which the phytochemicals of S. costus can be extracted.

Various reports suggest the phytochemicals extracted from S. costus such as costunolide, dehydrocostuslactone, and costic acid are effective in the treatment of numerous types of cancers such as lung cancer, prostate cancer, oral cancer as well as gastric cancer (Hunget al., 2010), (Kimet al., 2008; Tianet al., 2017), (Moonet al., 2013), (S. G. Koet al., 2004). When a human cell line was exposed to certain phytochemicals such as costunolide, dehydrocostuslactone, etc. tumor necrosis factor-alpha and interferon-gamma were activated, Alantolactone as well as caryophyllene were employed as treatments for the human keratinocyte cell line HaCaT (Rathoreet al., 2021). Costus causes fluctuations in the tumor suppressor genes and helps in restraining the growth of human breast, and liver cancer cells, etc. (Shatiet al., 2020). The cytotoxic effect of chloroformic extracts from S. costus was studied on various cancer cell lines such as lung cancer, Breast cancer, and colon cancer, it has been seen that effect of the extracts of the plant had a similar effect as that of the standard drugs such as doxorubicin provided to the patients suffering from breast cancer while no significant effect of the drug was observed on other cell line (Sunkaraet al., 2010). Shikokiols, an extract of S. costus has also shown anticancerous activities on various types of cancer cells as it arrests the cell cycle of the cancerous cells at the G2 phase and then induces apoptosis (S. G. Koet al., 2005).The sesquiterpene lactones are observed to possess anticancerous activity against prostate tumors (Tianet al., 2017). KB cells number was observed to be reduced when treated with IC₅₀ value (300 μg/mL) of S. costus extract as it reduces the growth of the cancer cells (Moonet al., 2013).

UL409 is an herbal formulation that contains S. costus extracts and is observed to possess anti-ulcer activity as it increases mucus discharge and improves gastric cell line protection (Gautam and Asrani, 2018). It has been reported (Sutaret al., 2011) that S. costus root extract when used in a particular dosage reduces the build-up of gastric acid and total acid. Still, it has also been noted that the effect was lower as compared to a standard drug.

Higher doses of S. costus extract suggested the possibility of humoral and cellular immunomodulatory activity in the immune system (R. S. Pandey, 2012). Costunolide, an extract of S. costus is observed to increase the levels of tyrosine phosphorylation in response to T-cell receptor cross-linking, according to research on the drug and dehydrocostus lactone (M. M. Pandeyet al., 2007; Taniguchiet al., 1995). KM1608 is an herbal formulation that contains the extracts of S. costus and is observed to induce immune-stimulatory effects in murine (Trinhet al., 2020).

According to the report submitted by (Negiet al., 2013), it has been observed that the costus oil can help in controlling diarrhea. It has been observed that when a group of Wistar rats induced with diarrhea were treated with castor oil, there was 26% to 67% inhibition in the diarrhea. There are a number of reports that claim the usage of costus for diarrhea and thus enhances its anti-diarrheal activity.

Costus is proven to be a safer and more organic option as a larvicide performed against A. albopictus (Rathoreet al., 2021) as it has been observed by (Liuet al., 2012), that certain extracts from the costus such as steroids, dehydrocostus lactone, and essential oil hinder the growth of the larvae of various insects, especially against A. albopictus, which transmits various diseases such as dengue fever. The larvicidal capacity of S. costus was confirmed by exposing the larvae to the methanolic root extracts of S. costus for 24 hr. and showed the best larvicidal activity with LC₅₀ and 90 values (Ali and Venkatesalu, 2020).

S. costus contains yellow -brownish oil extracted from its roots and it contains 39 components including dehydrocostus lactone and beta-costus as its significant constituents (Zaharaet al., 2014) hold significant value not just in the medical but other industrial sectors too. The S. costus oil has several benefits such as it can be formulated in hair oil, expensive perfumes, insect repellents, incense as it can blend with several other components such as vetiver, patchouli, rose, violet, and sandalwood (Rathoreet al., 2021). The costus oil composition depends upon the differences in the environmental condition, phenophase, ecotype, and chemotype as well as the genetics of the plant also influence the secondary metabolites of the plant (Rathoreet al., 2021). Other bioactive components present in the S. costus roots include sesquiterpenoids lignans, triterpenes, steroids, flavonoids, glycosides, etc.(Benedettoet al., 2019).

S. costus root oil contains sesquiterpene lactones in large concentrations which is responsible for various allergic contact dermatitis cases but still it holds high demand in perfumery (Gwariet al., 2013) because of its nice scent (Butola and Samant, 2010) but the presence of sesquiterpenoid α-methylene lactones, which is a harmful lactone can reduce the usage of S. costus root oil in perfumes (Rathoreet al., 2021).

There are seventeen chromosomes in all in S. costus (Singhet al., 2018), and despite having high medical importance, the exploration of the plant has not been done intensively, especially at the molecular level but through de novo leaf transcriptome analysis, the biosynthesis pathways of sesquiterpenoid and flavonoid found in S. costus were investigated. This helped to shed light on the plant’s metabolism and functional genomics (Rathoreet al., 2021).

Saussurea costus holds a significant value in the industry but due to various factors such as increased consumption, invasion of other species in the environment, deforestation, and some other factors like contamination of crops (Amaraet al., 2017) as well as heavy exploitation and an unscientific handling of the crop, its number has been significantly reduced, Thus The Red Data Handbook (RDB) of India and the International Union for Conservation of Nature and Natural Resources (IUCN) have declared S. costus as endangered and critically endangered, respectively(Siddiqueet al., 2001).

Additional factors contributing to the plant’s severely endangered status are the growing demand for it in the pharmaceutical business, the herb’s restricted distribution, and overuse. The Council of Scientific and Industrial Research (CSIR) initiated the CSIR Phytopharmaceutical Mission project (HCP 0010) to move the species from the critically endangered to the non-endangered category. This has resulted in the CSIR Institute of Himalayan Bioresource Technology, Palampur, HP, conducting extensive captive cultivation, and producing superior planting material (Rathoreet al., 2021).

Conventionally the plant is grown through the seeds but this approach is not suitable for the efficient growth of the crop because of lesser seed availability, and low percentage of germination, the cultivation of costus requires a patient approach as it takes up to three years to produce mature roots that are rich in valuable extracts like costus oil and other phytochemicals, This extended cultivation cycle is a significant challenge for plant growers, Therefore, micropropagation or in vitro cultivation of the plant becomes an important approach to for mass production of the S. costus (Sharmaet al., 2019).

Micropropagation is the process of vegetative development and multiplication of the plant from plant tissue that are artificially cultivated on nutritional media (Leifertet al., 1989). It is one of the main ways that contributes to the industrial cultivation of plants and has a lot of uses. (Jha, 2005). Micropropagation is based on the concept of omnipotence, which is the ability of plant cells and tissues to develop into whole new plants (Bhojwani and Razdan, 1996).

But this technique also includes the risk of somaclonal variance which can be encountered by analyzing the stability of the plant at various level of plant growth. The analysis can be performed at phenotypic, cytological, and phytochemical levels (Ghoshet al., 2002). To determine if the regenerated plant is genetically similar to its parent plant, chromosomal tests from tissue culture are necessary (Baran, 2005).