Nutrient dense Pigmented : A diet for Healthier People Overview

Rice is one of the most important crops in the world which is in great demand to feed more than half of the world's population and is being cultivated in a wide area from temperate to tropical zones. It is grown in more than a hundred countries, with a total harvested area of approximately 158 million hectares, producing more than 700 million tons annually. Nearly 640 million tons of rice is grown in Asia, representing 90% of global production. Sub-Saharan Africa produces about 19 million tons and Latin America some 25 million tons. In Asia and sub-Saharan Africa, almost all rice is grown on small farms of 0.5−3 ha.

In recent years, increasing the average life span and interest in correlation between food and human health has vigorously led to nutrigenomic studies. Their research outcomes has characterized the potential role of functional bio-actives in some specialty rice, known as pigmented rice.

Pigmented rice has a variety of colored grains like yellow, green, brown, red, purple and black. Due to rich nutritional landscape and antioxidant nutraceuticals, pigmented rice varieties have the potential to promote human health by managing various metabolic impairments. Due to poor awareness, yield ceiling, consumer preferences, staggered market potential, still it represents only 0.1% of total rice production. This bulletin provides information on Global and Indian scenario, the form of bioactive compounds, potential health benefits and a foreseen market for these niche rice varieties. The comprehensive overview also mentions the existing challenges and steps to be taken in this direction. Global scenario Rice is the most widely consumed staple food in the world and the world’s largest producers by far are China and followed by Indonesia, Bangladesh, Vietnam, Myanmar and Thailand. 41 Two species of domesticated rice exist : the Afghanistan widely cultivated Asian rice ( L.), 367 3011 domesticated in China some 10,000 years ago, Bhutan Bangladesh and the lesser-known African rice (Oryza 2826 glaberrima S.), domesticated about 3,000 years India ago. 1251 1821 Realizing the therapeutic potential and global Nepal Sri Lanka

demand, various countries explored their 797 pigmented rice and developed newer varieties. Pakistan In European countries, Italy and France, the red and black grain varieties of rice with a high content of phytochemical elements have been bred based on Asian varieties. Comprehensive evaluation of traditional rice collections (pigmented & non-pigmented) investigated mainly in South Asian countries like Afghanistan, Bangladesh, Bhutan, India, Nepal, Traditional rice varieties in IRRI gene bank Pakistan, Sri Lanka and deposited in IRRI rice gene bank. Pigmented rice varieties developed, collected and characterized globally Time Varieties Developed/ Countries Project period Collected/Characterized

Russian Federation State Developed by breeding- Russia 2012 Register of Protected Breeding Mars , Rubin (red-grained)

Russian Federation State Developed by breeding - Russia 2014 Register of Protected Breeding Yuzhnaya Noch (black-grained)

Heirloom Rice Collected -10114 Philippines 2005-2014 Project traditional varieties

Characterized - for cell wall UK- Enhanced Rice quality 2016-2018 polysaccharides and their contribution Philippines for Health (EnRicH) towards glycemic profile

National Natural Science Developed by genetic engineering China 2017 Foundation of China - purple endosperm rice “Zijingmii” Indian scenario India is home to 6000 rice varieties at present. Originally, India had more than 1,10,000 varieties of rice until 1970, which were replaced by the high yielding semi dwarf cultivars during green revolution with its emphasis on monoculture and hybrid crops. Diversity of pigmented rice, known from time immemorial for its nutraceutical benefits and remained as niche rice in various pockets of this country. Most well known are the of Manipur- Chak-hao Amubi, Chak-hao Poireiton and Chak-hao Angangba. Given below is a pigmented rice map of India based on existing scientific evidence available till date.

Pigmented rice map of India

1 [Jammu & Kashmir] Mirzag, Zag, Khuch, Khuch niver, Niver, Mirsagi, Zager, kawkreer, GS10, GS44, GS51, GS52, GS80, GS83) 2 [Himachal Pradesh] Chhohartu, Jatu, Matali, Karad, Sukara, Katheri, Begumi 3 [Arunachal Pradesh] Lingkang taker ame, Umling ame 4 [Manipur] Chakhao Poireiton, Chakhao Amubi, Wairi Chakhao, Khurkhul Chakhao, Pong Chakhao Chakhao Sempak, Chakhao Anganba and Langphou Chakhao 5 [Assam] Bairing Gurmu, Joradhan, Miren, Maibee, Maichukik, Dimrou, IRAT-141, Bairing, Sakbothung, Buarcha, Sakcharap, Vijoy Bijor and Pakai, Amana Bao,Betu, Biroi, Bogaguni,burali, Dal Bao, Hurupi Bao,Ixojoy, Jul bao, Kenkua bao, kolaguni, kotia Bao, Negheri Bao,Ronga Chokua, Ronga Dariya 6 [Jharkhand] Gora 7 [West Bengal] Kalabiroin, Mamihunger, Annapurna, Kalajeera 8 [Chattisgarh] Dimrou, IRAT-141,Bairing, Sakbothung, Buarcha, Sakcharap, Bairing gurmu Joradhan, Miren, Maibee, Maichukik 9 [Odisha] Kalabati, Keonjhar Kalachampa, Bhatamalli, Bhatachudi, Paradhan, Kandulakathi, Tikichudi, Beda Gurumukhi, Haladiganthi, Malligoindi, Kalachudi, Mallimakada 10 [Maharashtra] Kalabhat 11 [Telangana] Kakirekkalu, Burma black 12 [Karnataka] Kakka, Mappulai samba, Kusum kesar, Rajamudi rice 13 [Tamil Nadu] Vaigunda, Neelam samba, Kala samba, TKM9, Karungkuravai, red Kavuni, black kavuni, Koliyal, Kuruvi kar, Poongar, Kattu Yanam 14 [Kerala] Rakthashali, Njavara, Jyothi, Matta, MO1, MO5, Kari kagga, Matta triveni, Ptb1, 16, 17, 22, 23, 25, 29, 30 (Hraswa, Kattamodan, Karuthamodan, chuvannamodan, Kairali, Kanchana, Karthika, Aruna, Makom, Remanika, Revathy, Krishnanjana, Bhagya, Onam, Ahalya 3, Harsha, Varsha, Kunjukunju Varna, Kunjukunju Priya, Chingam, Sabari, Bharathy, Aathira, Aishwarya, Bhadra, Asha, Pavizham, Remya, Kanakom, Renjini, Pavithra, Panchami, Uma, Karishma, Vyttila-1,2,3,4,5, Arathy, Nila, Dhanya, Microstructure of pigmented rice

Over the decades, white grained rice varieties got preferred and the wild growing ancestors having colored grain have been only grown and consumed due to its cultural and therapeutic significance. Histochemical characterizations of pigments reported commonly in the pericarp, while few reports also suggest the presence in the endosperm. Black and purple rice have found with the presence of anthocyanins and their production is controlled by a promoter-diverged allele of OsB2, known as Kala4. Similarly, OsRc/Rd (Red pericarp, a homolog of OsB1/OsB2) regulates the pro-anthocyanidin production in the pericarp of red-grained rice. The thickness of pericarp varies among the rice genotypes mainly according to the microstructure (cellular layers), which also determines the quantum of accumulation of pigments.

(a) (b)

Pigment accumulation (a) in pericarp (b) both in pericarp & endosperm (adapted from Zhu et al., 2017,Molecular Plant 10, 918–929)

(a) black rice: venere (b) : ribe Microstructure of rice depicting pro-anthocyanidins (also known as condensed tannins) [red arrows shows pericarp; cdt-condensed tannins; adapted from Zaupa et al., 2016 Food structure (1): 6-12] Nutritional landscape of Pigmented rice Colored rice varieties are rich in pigments which are majorly antioxidants or functional bio-actives. The temporal expression of pigments during kernel development [till 40 Days after flowering (DAF)] has observed to start after 15DAF. Exclusively they are rich in polyphenols (250-1075 mg/100g) with having high free radical scavenging ability (69-93% by DPPH assay). Most abundant phenolics in black/purple rice are anthocyanins; while pro-anthocyanidins are predominant in red/ varieties. Other than bio-actives, pigmented rice is also reported to have higher contents of total dietary fiber, Changes in expression of pigments in black rice during kernel development. vitamin B1 and B 2 compared to white rice. The major A : 1 DAF, B : 3 DAF, C : 5 DAF, D : 7 DAF, fatty acids of pigmented rice are palmitic acid, oleic E : 9 DAF, F : 11 DAF, G : 15 DAF, H : 30 acid and linoleic acid. DAF, I : 40 DAF [Lee et al., 2012. Korean J. Crop Sci. 57(2): 127-131] Metabolic fingerprinting revealed the exclusive and predominant bio-actives present in pigmented rice

Bio-actives present in pigmented rice

Exclusively present Predominantly present Tocopherols Sterols tocotrienols oryzanol tocopherols sitosterol Flavones/ stigmasterol Anthocyanins Phenolic flavanols campesterol acids cyanidin-3-glucoside phytosterol apigenin peonidin-3-glucoside gallic acid kaempferol Others peonidin-3-O-rutinoside Carotenoids ferulic acid quercetin delphinidin-3-O-glucoside sinapic acid inositol isorhamnetin zeaxanthin cyanidin 3-O-sophoroside folic acid Pro-antho- beta carotene ellagic acid cyanidin 3,5-O -diglucoside α-lipoic acid cyanidins lutein catechuic acid cyanidin 3-O-rutinoside pangamic acid keracyanin (C3rutinoside0Cl) catechin phytic acid malvidin glucoside epicatechins pro-anthocyanidin dimers [Partially adapted from Kusano et al., pro-anthocyanidin trimers 2015 Molecular Plant 8(1):58-67] Potential health endorsements Anti-oxidant effect Oxidative stress (OS), the major hidden reason behind various disease etiologies occurs when production of reactive oxygen species (ROS) overwhelms the inherent antioxidant system. Such OS leads to cellular damage which end in apoptosis. Such redox imbalance kick starts a cascade of genomic, epigenomic, transcriptomic, proteomic as well as metabolic factors that leads to inflammation associated cell death. Pigmented rice rich in antioxidants like anthocyanins, pro-anthocyanidins, tocopherols, carotenoids etc, efficiently chelate the free radicals which are the hidden reasons behind life style disorders.

Role of antioxidant in preventing/managing ROS induced metabolic alterations

Cellular protein Oxidation

Nitro tyrosine, carbonyls

Cellular lipid Oxidative Cellular DNA Peroxidation burst Oxidation

Isoprostanes, hydroxyl guanine, Hydroxy-nonenan nitroguanine

Cellular Glycoxidation

AGEs, glycolipids, glycoproteins

Nitrate radical Cellular oxidative burst scavenging by anthocyanins & potential markers Anti-obesity effect

Pigmented rice has proved to have effect in weight reduction as it contains higher fiber and less digestible starch content, which assists to stop signals of hunger and discourage excessive consumption. Oxidative stress induced inflammation being key signal during obese condition and antioxidant rich pigmented rice has possible role in its alleviation. Supplementing with pigmented rice/extracts significantly reduced body weight gain and lipid accumulation in the liver and adipose tissue in animal models as well as in clinical trials. Triglyceride, total cholesterol, and low- density lipoprotein-cholesterol levels in serum were decreased by supplementation, whereas high-density lipoprotein-cholesterol level significantly increased. This, in turn, decreased PPARγ, SREBP-1c, FASN, ACC, and HMGCOR-A and increased LXRα, PPARα, and CPT1α at the gene level. Further, it lowered PPARγ and elevated PPARα, p-ACC/ACC, and p-AMPKα/AMPKα at the protein level. Additionally, treatment decreased gene expression of NPC1L1, ACAT2, MTTP, which regulated lipid absorption in the intestine and preserved CYP7A1, ABCG5/8 that transported cholesterol from the liver and enterocytes to the intestinal lumen.

(Adapted from Liu et al., 2019 Journal of Functional Foods 3:17-32) Anti-cancer effect Increasing evidence from animal, epidemiological and clinical investigations suggest that dietary anthocyanins/phenolic acids have potential to prevent chronic diseases, including cancers. Anti-metastasis potential of black rice anthocyanins through human epidermal growth factor receptor 2 (ErbB2) has been established. Recent work demonstrates that pigmented rice bran extracts are helpful in attenuating inflammation and inflammation induced cancer progression.

Infiltration of inflammatory cells

Secretion of pro-inflammatory cytokines (Interleukin 6, Tumor Necrosis Factor (TNF)-α ) Anti-metastasis effect*

Other general health benefits* Immune health Nutritional psychiatry is a growing discipline that focuses on the use of Boosts immunity, food and supplements to provide these quench stress induced essential nutrients as part of an free radicals; High Fe & integrated or alternative treatment for Mn content. mental health disorders. Pigmented Anti-allergic rice being rich source of antioxidant cocktail, could play role in this which will Black rice is broadly known as enriched rice be beneficial for future generations. and has been used in traditional medicine. Being gluten free, it is suitable for celiac patients also. Digestive health

The dietary fiber per serving is remarkable in pigmented rice. Among which insoluble fiber enables to protect against constipation, bloating and other undesirable digestive symptoms. While soluble fiber like resistant starch act as Detoxification prebiotic for healthy gut microbiome.

Consumption of pigmented rice can detoxify the body and purify the liver with the use of elevated antioxidant levels in the rice. The anti-oxidative status of blood, liver, and the aorta assessed resulted with less oxidative stress.

* Samyor et al., 2017 International Journal of Food Science and Technology doi:10.1111/ijfs.13378 Anti-diabetic effect

Asians are known to have a genetic predisposition to poor insulin secretion by pancreatic β-cells and their insulin secretion is prone to decline with β-cell exhaustion. Pigmented rice rich in spectrum of antioxidants like anthocyanins have thus been studied for its anti-diabetic effect using in vitro & in vivo models. A significant reduction in glycosylated hemoglobin, fasting glucose, and improvements in serum insulin level has been observed in mice models treated with pigmented rice extracts. Anthocyanins have deciphered to play pivotal role in regulating glucose uptake in the intestine and muscle via regulating the secretion of insulin from the pancreas through modulating glucose transporter (GLUT) receptors, thus bestowing the anti-diabetic effect.

Glucogenolysis Glucagon Gluconeogenesis During fasting blood glucose levels Glycogenesis

Liver Gluconeogenesis Normal blood Insulin glucose Glucose uptake levels 4.4-6.1 mM or Adipose After meals 80-125mg/dL blood glucose Glucose uptake levels

Glucose Muscle

Figure depicts the role of pigmented rice in regulating blood glucose homeostasis through inter-organ communication. Pancreas regulates glucose using insulin and glucagon in response to food intake. Further Insulin also acts on peripheral tissues (liver, adipose tissue and skeletal muscles) to increase glucose uptake, resulting in decreased blood glucose levels. [Green lines denotes the normal glucose response and red lines denotes the response during hyperglycemic conditions]. Role of “Anti-aging & managing glucose homeostasis”

Red rice was characterized by a high quantity of oligomeric procyanidins (0.2 mg/g) with more than 60% of total phytochemicals found in the rice seeds. Procyanidins are high molecular weight polymers or complex flavan-3-ol polymers that consist mainly of catechin, epicatechin, gallocatechin, and epigallocatechin units that can also be found in rice germ and bran.

Treated with red rice phenolics

Intracellular glucose (C14 ) uptake Glycation potential Expression of GLUT receptors & gluconeogenic genes HepG2 cells (in vitro hepatocyte model)

Glycation Damage: A hub for aging & associated disorders

Figure depicts the role of red rice phenolics in managing glucose homeostasis using in vitro assays. Various parameters like C14 Glucose uptake, expression of GLUT receptors and gluconeogenic genes were validated. The in vitro protein glycation assay developed would also assist in aging studies in future Role of black rice “Anthocyanins modulate oxidative stress” Major antioxidant, anthocyanins/anthocyanidins modulate oxidative stress by chelating free radicals. More than 500 different anthocyanins and 23 anthocyanidins have been yet reported. Anthocyanins exist as mono-, di-, or tri-O-linked glycosides and acyl glycosides of anthocyanidins in pigmented rice. The sugar moiety may be substituted by aliphatic, hydroxybenzoic, or hydroxycinnamic acids. Major forms are cyanidin 3-O-glucoside (2.8 mg/g) and peonidin-3-O-glucoside (0.5 mg/g).

In vivo hyperglycemic mouse model

Rice powder extract Oral gavage (200mg/kg) Serum & Pancreatic histopathology Tissue collection

Figure depicts the histological effects on pancreatic beta islets due to Streptozotocin (STZ) induced hyperglycemia in mouse model by Haematoxylin and Eosin staining (WR-White rice; BR-Black rice & RR-Red rice; E - extract) Abnormal histological atrophy of pancreatic beta islets due to Streptozotocin (STZ) induced hyperglycemia was drastically reduced when supplemented with pigmented rice. Restoration of pancreatic islets in number and size was observed. Anthocyanin profiles using UPLC Work carried out at Division of Biochemistry, ICAR-Indian Agricultural Research Institute (IARI) in collaboration with CSIR-Indian Institute of Chemical Biology (IICB), Kolkata The predominant anthocyanins prevent the oxidative stress induced damage of pancreatic islets Market potential

Presence of health plus contents gave pigment rice ‘super food’ status and thus fetching premium price (Rs.300-500/Kg) Therapeutic effects of anthocyanins has boomed the fortification industry where varied percentage of anthocyanin has been fortified (2 - 25%) in bread, confectionaries, desserts etc. Black rice farmers are hugely benefitted with the market potential of crude and pure black rice powder which are used as natural colorant (E163) as well as for supplements.

Black rice powder (5-25% anthocyanin) Purified anthocyanin (E163) Black rice - Rs. 300-500/Kg Rs.15,000-20,000/Kg Rs.30,000-50,000/Kg

The global anthocyanins market was valued at USD 291.7 million in 2014 and is expected to reach USD 387.4 million by 2021, growing at a CAGR of 4.4% from 2015 to 2021 Conclusion Pigmented rice is a miracle food and can be a panacea of maladies. The bioactive compounds exclusively and predominantly present in pigmented rice display significant potential with regard to a range of beneficial health effects. There is also the prospect of additional practical implications, not only for agriculture expansion but in the food industry as well. Poor consumer preferences due to palatability issues (eating and cooking qualities) being major limitation, breeding/genetic engineering efforts are need of the hour to escalate the status of this wonder grain. Several pigmented rice varieties have been used to extract nutraceuticals, and these seem to hold a promise in terms of potential cosmeceutical utilization in the new global business era. But currently this is handicapped by lack of efficient high throughput extraction methods, which have to be tackled. Emerging domains of nutritional psychiatry hold new promises, not only for the present generation but also for the future which underlines the importance of health plus compounds in pigmented rice. February, 2020

Citation: Veda Krishnan,1 Archana Singh, 1 Susheel Sharma, 2 Haritha Bollinedi, 3 U Mabalirajan4 , Suneetha Kota 5 , Sandeep Raja 6 , A.K Singh 3 and Shelly Praveen 1 (2020) Nutrient dense pigmented rice: A diet for healthier people 1. Division of Biochemistry, ICAR-Indian Agricultural Research Institute (IARI), New Delhi 2. ICAR – Research Complex for NEH Region, Manipur Centre, Imphal 3. Division of Genetics, ICAR-Indian Agricultural Research Institute (IARI), New Delhi 4. CSIR- Indian Institute of Chemical Biology, Kolkata 5. ICAR- Indian Institute of Rice Research, Telangana 6. Acharya N.G. Ranga Agricultural University, Telangana

TB-ICN:236/2019 Published by Division of Biochemistry ICAR-Indian Agricultural Research Institute New Delhi