Pharmacological and analytical aspects of alkannin/shikonin and their derivatives: An update from 2008 to 2022

Alkannin/shikonin (A/S) and their derivatives are naturally occurring naphthoquinones majorly found in Boraginaceae family plants. They are integral constituents of traditional Chinese medicine Zicao (roots of Lithospermum erythrorhizon). In last two decades significant increase in pharmacological investigations on alkannin/shikonin and their derivatives has been reported that resulted in discovery of their novel mechanisms in various diseases and disorders. This review throws light on recently conducted pharmacological investigations on alkannin/shikonin and their derivatives and their outputs. Various analytical aspects are also discussed and brief summary of patent applications on inventions containing alkannin/shikonin and its derivatives is also provided.

In the time frame of 1969 to 2021, a total of 634 full text reports are available in PubMed database and out of these, and 606 reports are published after 2000, showing the increased interest of research groups in A/S and their derivatives. Trend analysis suggests that researchers are more focused on shikonin than alkannin (Fig. 3). An exhaustive review of A/S and their derivatives was first published by Papageorgiou group in 1999 (Papageorgiou, Assimopoulou, Couladouros, Hepworth, & Nicolaou, 1999). A decade later, another update was published with prime focus on wound healing and associated bioactivities (Papageorgiou, Assimopoulou, & Ballis, 2008). In 2013, Andujar group published a compilation containing pharmacological investigations on A/S and their derivatives for the period of 2002 to 2013 (Andújar, Ríos, Giner, & Recio, 2013). Subsequently, various review reports were published by different research groups with a focus on either individual bioactivity or on individual derivative. A/S and their derivatives possess enantiomeric properties that make their analysis quite complex. Surface-enhanced Raman Spectroscopy (SERS) and chiral HPLC have been successfully utilized for differentiating A/S and their derivatives (Cañamares, et al., 2022;Azuma et al., 2016). Literature analysis suggests that after 2008 (Papageorgiou, Assimopoulou, & Ballis, 2008), any review update regarding analytical aspects of A/S and their derivatives is not available. Thus, there is a dire need of an updated compilation containing all pharmacological, analytical and miscellaneous investigations on A/S and their derivatives. Forecasting the marketable potential of A/S and their derivatives, wide range of patents have been filed by various research groups around the globe for various applications to safeguard their usage. This review is primarily focused on providing update on various investigations on A/S and their derivatives from year 2008 to 2021 along with thorough insight on the patent applications filed.

Wound healing activity
Dried roots of Arnebia guttata Bung, Arnebia euchroma (Royle) Johnston, and Lithospermum erythrorhizon Sieb. Et Zucc loaded oil based ointment (Zicao) has been widely used for treatment of wounds (Chak, Hsiao, & Chen, 2013;Hsiao, Tsai, & Chak, 2012;Lu et al., 2008;Zeng & Zhu, 2014). The major active components of Zicao include shikonin and its derivatives such as deoxyshikonin, acetylshikonin and b,b'-dimethylacrylshikonin. Furthermore, to overcome the demerits of this oil based ointments such as discomfort, irritation and difficulty in cleaning, soluble water based topical preparation such as Zicao-HP-b-CD complex was formulated using 2-hydroxypropyl-b-cyclodextrin to form watersoluble complex which resulted in its enhanced bioavailability and stability. The active ingredients of Zicao enhance collagen synthesis in granuloma tissues and promote inactivation of tumor necrosis factor-a gene expression (Chen, Yu, Hsu, Tsai, & Tsai, 2018). On the other hand, Jawoongo, a Korean traditional medicine has been found highly effective in removing necrotic tissue caused by burn wounds. Jawoongo consists of Lithospermi Radix, Angelicae Gigantis Radix, Ronicerae Flos, Glycyrrhizae Radix, Coptidis Rhizome and Scutellariae Radix. The major active ingredient is Lithospermi Radix which mainly comprises of deoxyshikonin. It significantly increases the phosphorylation of p38 and ERK1/2 in a concentration dependent manner. Additionally, it activates Mitogenactivated protein kinase (MAPK) signaling which promotes cellular migration and angiogenesis. It was observed that deoxyshikonin induced migration and proliferation in HaCaT cells mediated through activation of p38 and ERK respectively. Thus, the study demonstrated that deoxyshikonin possesses strong ability for proliferation, migration and tube formation of HaCaT and HUVEC cells, which in turn promotes angiogenesis (Kim, Lee, & Yook, 2013;Park et al., 2017).
Recently, an increased attention is focused on the herbal medicines attributing to their quality, safety and efficacy. Since ancient times, people have used plant based preparations to promote wound healing process (Fronza, Heinzmann, Hamburger, Laufer, & Merfort, 2009). Various plants especially belonging to Boraginaceae family have been reported to possess excellent therapeutic potential in wound management. The main active metabolites of this family are naphthoquinones which possess antiinflammatory, anti-microbial, anti-oxidant activities contributing to wound healing (Lee et al., 2016). Meanwhile, additional studies demonstrated that therapeutic benefits of roots of Boraginaceae family plants are wider than its aerial parts. The most active components found in roots are shikonin, alkannin, deoxyshikonin and acetylshikonin. Traditionally, the root extract of Onosma dichroantha Boiss. has been used in Iran for healing burn wounds. Furthermore, the cyclohexane fraction has been found to be most potent inhibitor of lipopolysaccharide induced nitrogen oxide production which accelerates fibroblast proliferation, tissue regeneration and angiogenesis. Active components present in the cyclohexane fraction were found to be shikonin, arnebin-1 and b,b'-dimethyl acrylalkannin. Among all of these components, arnebin-1 has proangiogenic and synergistic effects with vascular endothelial growth factor (VEGF) which further augments the wound healing process (Safavi et al., 2019). Similarly, several other phytoconstituents isolated from n-hexane-dichloromethane extract of Onosma argentatum Hub.-Mor. roots i.e. deoxyshikonin, acetyl- K. Kaur, R. Sharma, A. Singh et al. Chinese Herbal Medicines 14 (2022) 511-527 shikonin, 3-hydroxyisovalerylshikonin and 5-8-Odimethylacetylshikonin were found to be effective in treatment of burns wounds. In another study, the efficacy of mixture of olive oil, beeswax and root extract of Alkanna tinctoria Tausch. was examined on burn wounds which showed rapid epithelization and angiogenesis (Gümüs ß & Özlü, 2017). Moreover, this extract has been established to increase fibroblasts production which amplifies tissue regeneration and provides better perfusion to wound area resulting in granulation tissue formation (Yazdinezhad, Monsef-Esfahani, & Ghahremani, 2013) (Fig. 4). The healing effects of ointment loaded with Arnebia euchroma extract were also compared with standard silver sulfadiazine on second degree burns and the extract demonstrated higher efficacy. Fibroblast proliferation, cell migration and collagen synthesis were observed to be the major mechanisms in its healing process (Nasiri et al., 2016). K. Kaur, R. Sharma, A. Singh et al. Chinese Herbal Medicines 14 (2022) 511-527 Furthermore, the active constituents of L. erythrorhizon such as shikonin, isobutyl-shikonin, b-hydroxyl-isovaleryl-shikonin and amethyl-n-butyl-shikonin were loaded in chitosan/gelatin-based scaffolds and examined for their wound healing potential. The results demonstrated the mechanism of healing via regulation of epithelial-mesenchymal transition (EMT) through TGF-b expression (Table 1) (Hsiao, Tsai, & Chak, 2012;Wang, Kravchuk, & Kimble, 2010;Yao, Chen, Chen, Li, & Huang, 2019).

Antimicrobial activity
Traditional Chinese herb L. erythrorhizon has been widely used in treatment of a wide range of infections (Yan, Tan, Miao, Wang, & Cao, 2019). Candida albicans is the major opportunistic pathogen and major cause of fungal infections in humans. Shikonin showed significant inhibitory effect on the growth of C. albicans through multiple mechanisms. It markedly increases the intracellular ROS (reactive oxygen species) and causes depolarization of mitochondrial membrane potential. It was observed to reduce the ergosterol content also. Further, it could lead to the upregulation of thioredoxin reductase-related gene (TRR1), NADPH oxidoreductase-related gene (EBP1) and mitochondrial respiratory electron transport chain-related gene (MRF1) (Fig. 5) (Miao et al., 2012). Moreover, shifting mitochondrial aerobic respiration and promoting endogenous reactive oxygen species augmentation contributes to DNA damage (Liao et al., 2016). On the other hand, Staphylococcus aureus is one of the most common and predominant causes of persistent infections in chronic wounds; It contributes the nosocomial infections and hence, proved to be biggest pathogenic burden. Being an adaptable pathogen, it has ability to gain resistance against broad spectrum of antibiotics. Resistance development occurs as a result of horizontal gene transfer (HGT) via transduction, conjugation, or transformation (Craft, Nguyen, Berg, & Townsend, 2019). However, resistance to methicillin and other b-lactam antibiotics is acquired due to mecA gene transfer situated on a mobile genomic element, the Staphylococcal chromosome cassette mec (SCCmec) (Deurenberg & Stobberingh, 2009). In order to combat multidrug resistance, shikonin is of utmost importance. The TEM images of shikonin treated Methicillin-resistant Staphylococcus aureus (MRSA) shows disruption of cytoplasmic membrane and cell lysis with subsequent leakage of intracellular components. In addition, shikonin directly binds to peptidoglycan (PGN) which is main component of Gram-positive bacterial cell wall (Lee et al., 2015).
Streptococcus pneumoniae is another pathogen causing severe infections in humans. Pneumolysin (PLY) is vital virulence trait of S. pneumoniae which possesses cytotoxicity, genotoxicity and pro-inflammatory activity. The treatments for this microbe were limited due to its ubiquitous antibiotic resistance. Shikonin has been found as therapeutically effective for S. pneumoniae based pneumonia as it antagonizes the hemolytic activity of PLY thereby reducing the cytotoxicity of PLY. It also inhibits oligomers formation and block pore formation on the cell membrane which leads to decreased production of IFN-c and IL-6 . Moreover, shikonin was also found effective for periodontal diseases as it has ability to inhibit Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mutans and Lactobacillus acidophilus which are most susceptible bacterial strains involved in dental caries (Table 2) (Li, Xu, Zhu, & Wang, 2012). In the latest studies, shikonin and its derivatives including shikonin glucoside, 4chlorophenylacetyl shikonin, lithospermidin B and Angelyl shikonin were assessed for protein binding with Main protease (Mpro) of SARS CoV-2 revealed shikonin and some derivatives as potential antiviral agent of Covid (Woo & Das, 2022).

Anticancer activity
Cancer is one of the most fatal diseases and one of the primary causes of deaths globally. The incidence of cancer in India has been expanding in the last two decades as in other developing nations. Not only the incidence but pattern has also changed to a great  extent (Ferlay et al., 2010;Jha, 2009;Guddati, 2012;Rocconi et al., 2012). Anticancer drug resistance is another major obstacle in the effective cancer treatment. It is known that conventional anticancer drugs are likely to cause apoptosis. Due to sensitivity to neoplastic cells to apoptosis, they significantly become resistant via antiapoptotic progression and dysregulation of apoptotic machinery (Han et al., 2007). In addition, antiapoptotic progression in neoplastic cells involves overexpression of antiapoptotic proteins (Bcl-2, Bcl-x1, Mcl-1, c-FLIP), proapoptotic proteins mutations (p53, Apaf-1, Bax, FAS) and loss of caspases (Caspase-3 and Caspase-8) which significantly contributes to drug resistance (Bonora et al., 2015). Therefore, defects in the apoptotic signaling and upregulation of apoptotic inducers enormously limit the effectiveness of chemotherapy. Presently, overcoming the drug transporter-mediated resistance is possible as it works on fewer targets whereas apoptosis mediated drug resistance is highly difficult because of multiple potential targets (Han et al., 2007). Owing to its strong and broad spectrum anti-cancer activity, shikonin and its derivatives are gaining popularity. A study by a research group revealed the necroptotic mechanism of shikonin to promote non-apoptotic cell death (Degterev et al., 2005). Moreover, shikonin could circumvent cancer drug resistance through induction of necroptosis. Necroptosis is a programmed cell death characterized by necrotic cell morphology and activation of autophagy (Han et al., 2007). Also, shikonin promotes topoisomerase mediated DNA cleavage, caspase-dependent apoptosis and cell cycle arrest via activation of tumor suppressor gene p73 and downregulation of ICBP90 (Fig. 6). Additionally, p73 is responsible for transcription of various p53 target genes such as p16 INK4A , PIUMA (p53-upregulated modulator of cell death) and p21 (Jang, Hong, Jeong, & Kim, 2015).
A recent report indicates that ICBP90 is overexpressed in patients with cervical cancer. Cervical cancer is second most malignant tumor in women after breast cancer. Annually, the global rate of cervical cancers is about 60 million cases with 25 million deaths (Kaarthigeyan, 2012). Also, high risk human papillomavirus infection (HPV) long term infection of HPV is the leading cause of cervical intraepithelial neoplasia, precancerous lesions and cervical Murine macrophages (RAW264.7), normal human skin fibroblasts (Hs27), human microvascular endothelial cells (HMEC-1), zebrafish line TG (fli1: EGFP) Inhibition of LPS-induced NO production thereby promoting tissue regeneration and angiogenesis Burn wounds Safavi et al., 2019 Alkannin, shikonin, juglone, a- Human dermal scar-derived fibroblasts (HSF) and Human 'normal' dermal fibroblasts (nsHSF) Inhibition of TGF-b1 induced collagen deposition and cell mediated contraction; Phosphorylation of P-Erk and NF-jb  carcinoma (Cook et al., 2017). Previous studies demonstrated that b-hydroxyisovaleryl shikonin (b-HIVS), a shikonin derivative, possesses inhibitory effect on HeLa cells through apoptosis and prevent tumor cell proliferation. b-HIVS retards PI3K activity and downregulates AKT/mTOR signaling along with reduced P7056K expression levels which ultimately leads to tumor suppression (Lu et al., 2015). On the other side, breast cancer is most prevalent malignancy in women. Recently, triple-negative breast cancer (TNBC) accounts for about 20% of all new cases of breast cancer accompanied with higher grade and distinct metastatic potential. Therefore, suppression of metastasis might be a promising therapy for TNBC patients (Lambert, Pattabiraman, & Weinberg, 2017;Temian, Pop, Irimie, & Berindan-Neagoe, 2018). Essentially, epithelial-to-mesenchymal transition (EMT) plays a pivotal role in regulating metastasis process. EMT involves loss of epithelial phenotypes and the gain of mesenchymal features. It is characterized by downregulation of epithelial cell-surface markers such as occludin, E-cadherin and zonula occludens-1 whereas upregulation of mesenchymal markers such as N-cadherin and vimentin. Of particular interest, shikonin has been established as an effective strategy with good therapeutic potential for TNBC patients. It significantly reduces the expression of miR-17-5p which leads to activation of tumor suppressor gene (PTEN). However, overexpression of PTEN downregulates the Akt expression thereby inhibiting metastasis (Bao et al., 2020).
In recent years, the incidence of colon cancer is considerably increasing in western countries attributing to unhealthy lifestyles. The safety and efficacy of shikonin was determined against colon cancer. Studies demonstrated that shikonin promotes cell death via mitochondrial dysfunction which is induced by downregulation of Bcl-2 and upregulation of Bax, Caspase-3 and Caspase-9. In addition, activation of MAPK pathway and increased endoplasmic reticulum stress triggers apoptosis (Han et al., 2019;Liang et al., 2017). Specifically, anti-cancer activity of shikonin against gefitinib-resistant non-small cell lung cancer (NSCLC) was investigated. Shikonin showed strong cytotoxicity against NSCLC cell lines. Also, it effectively generates ROS and stimulates EGFR degra-dation resulting in inhibition of TrxR thereby inducing apoptosis . Another study on paclitaxel-resistant non-small cell lung cancer, shikonin induces dysregulation of NEAT1 expression which leads to deactivation of PI3K/Akt pathway hence, inhibiting cell proliferation. Simultaneously, shikonin considerably increases expression of PARP and caspase-3 and caspase 9 cleavages (Zang, Rao, Zhu, Wu, & Jiang, 2020). Researchers reported that activation of STAT3 and PKM2 regulates cell proliferation (Cao et al., 2020;Hoshino, Hirst & Fujii, 2007). Therefore, STAT3 and PKM2 can be considered as key targets for tumor suppression. Recent studies indicated that shikonin markedly reduced the expression of STAT3dimer and PKM2 gene thereby inhibiting inhibits melanoma cell growth (Cao et al., 2020;. Furthermore, the deactivation of NFƙB also contributes in inhibiting cancer-inducing inflammation by decreasing release of inflammatory cytokines such as COX-2, iNOS and IL-6 ( Table 3). In the recent studies shikonin was tested against Acute Myeloid Leukemia. Shikonin impairs the mitochondrial activity and electron transport chain complex-II to selectively target leukemia cells (Roma et al., 2022). Moreover, inhibitory potential of shikonin was reported on Sunitinib-Resistant renal carcinoma cells. It acts by necrosome complex formation and downregulation of AKT/mTOR signaling pathway (Markowitsch et al., 2022). Lately, shikonin was tested and found effectively active against Mutant-non small lung cancer cells. It induces necrosis and apoptosis of cancer cells via thioredoxin reductase 1 inhibition following SecTRAPs generation and oxygen-coupled redox cycling pathway (Zhang et al., 2022). One of the study demonstrated anticancer effect of shikonin against colon cancer cells. It triggers the apoptosis of cancer cells by checking the cancer cell growth in S phase of cell cycle (Chen et al., 2021). Shikonin is found to be a potential inhibitor in pancreatic cancer as it mediates PD-L1 degradation which in turn suppresses immune evasion in pancreatic cancer cells via NF-jB/STAT3 and NF-jB/CSN5 signaling pathway (Ruan et al., 2021). The anticancer potential of shikonin co delivered with siTGF-b against triple negative breast cancer cells was investigated by Li et al and this codelivery approach was found to be magnificently efficacious for the same (Li et al., 2022). In a nutshell, shikonin/alkannin and their derivatives are promising candidates for anticancer activity which act by various signaling pathways.

Miscellaneous activities
Apart from pharmacological activities discussed above alkannin/shikonin and their derivatives also possess therapeutic potential against phytogenotoxicity, bronchial asthma, peptic ulcer, spasmogenicity, atherosclerosis, inflammatory diseases, ischemic heart diseases, cataract, hepatotoxicity and impotency (Fig. 7) (Yildirim, 2020). Onosma, the biggest genus of Boraginaceae family, is being used as traditional medicine since centuries (Davis, 1970). Shikonin and its derivatives have also been reported to inhibit oxidized low-density lipoprotein (LDL) induced monocyte adhesion by deactivation of NFƙb and hence used in treatment of atherosclerosis. It is well known that oxidized LDL plays a key role in thrombosis, endothelium apoptosis and vascular smooth muscle proliferation. In addition, it also stimulates release of inflammatory mediators such as cytokines and reactive oxygen species. Moreover, activation of NFƙb further upregulates the expression of intracellular adhesion molecule (ICAM-1), E-selectin, vascular cell adhesion molecule and monocyte chemotactic protein-1. Hence, the accumulation of oxidized low-density lipoprotein (oxLDL) and inflammatory cells lead to atherosclerosis. Shikonin has also been found effective in retarding oxLDL mediated ROS production through induction of expression of PI3K/Akt/Nrf 2-dependent antioxidant genes such as SOD-1, HO-1, Catalase, GPx-1, GCLM, and GSR (Huang et al., 2015). Furthermore, the oxidative stress is the major cause of various other medical conditions such as ageing, diabetes, stroke, neurodegenerative disorders, cancer etc. Oxidative stress is often accompanied with higher blood sugar levels. The skeletal muscle cells are rich in insulin-sensitive glucose transporters named as glucose transporter 4 (GLUT4). Their main function is translocation of glucose from cytoplasm to cell membrane aiding in glucose uptake. Therefore, it plays imperative role in regulation of homeostasis of glucose. However, the contraction-induced release of reactive oxygen species (ROS) and activation of AMP activated protein kinase (AMPK) may also lead to increased glucose uptake in skeletal muscle cells (Mao, Yu, Li, & Li, 2008;Su, Huang, & Zhu, 2016). Subsequently, acetylshikonin-induced glucose uptake was significantly inhibited by reduction of PLC-b3 in L6 myotubes, which makes it evident that acetylshikonin-induced glucose uptake may be triggered by activation of inositol lipid signaling and increased DAG release . On the other hand, ageing is also considered as biggest cause of Alzheimer's disease. Various studies have shown that oxidative stress, neuronal apoptosis and neuroinflammation plays critical role in pathogenesis of Alzheimer's disease (Heneka, 2015). SIRT1 is essentially involved in cognitive functions and shows protective effect against aging-related neuronal degeneration. Thus, SIRT1 can be the most promising therapeutic target for Alzheimer's disease. Multiple studies reported that chronic inflammation associated with raised levels of pro-inflammatory mediators such as IL-6, IL-10, TNF-a and IL-1b. Notably, acetylshikonin reduced the levels of these mediators via inhibiting the activation of NFƙb and thereby reducing inflammation. Simultaneously, it also inhibits the activation of p21/p53 signaling pathway (Chang et al., 2015). Furthermore, overexpression of thymic stromal lymphopoietin (TSLP) is a major factor contributing to allergic diseases such as asthma, allergic rhinitis etc. Epithelial cell-derived TSLPs control the allergic condition via regulating the activation of T-cells, mast cells, and dendritic cells. The findings of the study elucidated that shikonin as well as L. erythrorhizon aqueous extract was able to downregulate TSLP production as well as markedly attenuated the levels of IKKa, NLRP3 and Caspase-1 (Yen et al., 2017). Besides having multiple pharmacological effects, naphthoquinones are also considered as potent allelochemicals as they hold good potential to defend against predators. Previous studies demonstrated that juglone, 1,4-naphthoquinone, plumbagin and 2-methoxy-1,4-naphthoquinone showed antifeedant activity against the cabbage looper Trichoplusia ni (Akhtar, Isman, Niehaus, Lee, & Lee, 2012). Napthoquinones were also found effective against the dry bean pests Epilachna varivestis and Acanthoscelides obtectus (Cespedes et al., 2016). Moreover, the extreme toxicity of juglone against Myzocallis walshii and plumbagin against Tetrany chusurticae, Myzus persicae and Illinoia liriodendri were also investigated. These studies substantiate that juglone and plumbagin are effective insecticidal and acaricidal agents. The inhibitory and toxicity potential of Onosma visianii roots against Spodoptera littoralis was also investigated. The main active constituents of O. visianii roots include isovalerylshikonin and isobutyrylshikonin. Being highly lipophilic in nature, these active moieties easily enter the insect exoskeleton and hinder the physiological processes. Moreover, the ester groups of these moieties increases cuticle penetration via linkage with hydroxyl groups and significantly inhibits acetylcholinesterase (AChE)     enzymes. Additionally, it leads to inhibition of mitochondrial respiration thereby controlling larval growth (Table 4) Pavela, 2013).

Patent applications
Forecasting the market potential, numerous patent applications on inventions containing alkannin/shikonin and its derivatives have been filed by various research groups across the world. Brief details of these applications are divided into two categories viz. therapeutic and analytical and are summarized in Tables 6 and 7.

Conclusion
Alkannin/shikonin and its derivatives possess a wide variety of pharmacological activities. These constituents are majorly investi- gated for their wound healing, antimicrobial and anticancer potential. In the last decade, various mechanisms of alkannin/shikonin and their derivatives are explored implicated in wide variety of diseases. The present study suggests the higher applicability of alkannin/shikonin and its derivatives are in the development of potent and safer wound healing and anticancer agents. Various analytical investigations are also discussed that will help the analysts for more efficient analysis of alkannin/shikonin and its derivatives from different sources. Brief patent summary is provided to highlight the future marketable potential of alkannin/shikonin and its derivatives. The appropriate knowledge of the pharmacological aspects of A/S and their derivatives will not only benefit the natural product researchers but also the pharmaceutical/formulation scientists in their future course of action. Further, the advanced and novel drug delivery systems could be used to mask the limitations of these derivatives including their low solubility and photo degradation. Despite having magnificent pharmacological potential, there is a dire need to collect remarkable data related to their toxicological and safety profile which can establish the clinical usage of these components.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.